Early Mammalian Development Research Articles

Genomewide decoupling of H2AK119ub1 and H3K27me3 in early mouse development

Polycomb group (PcG) proteins are crucial chromatin regulators during development. H2AK119ub1 (H2Aub) and H3K27me3 are catalyzed by Polycomb-repressive complex 1 and 2 (PRC1/2) respectively, and they largely overlap in the genome due to mutual recruitment of the two complexes. However, it is unclear whether PRC1/H2Aub and PRC2/H3K27me3 can also function independently. By developing an ultra-sensitive carrier-DNA-assisted chromatin immunoprecipitation sequencing method termed CATCH-Seq, we generated allelic H2Aub profiles in mouse gametes and early embryos. Our results revealed an unexpected genomewide decoupling of H2Aub and H3K27me3 in mouse preimplantation embryos, where H2Aub but not H3K27me3 was enriched at PcG targets while only H3K27me3 was deposited in the broad distal domains associated with DNA methylation-independent non-canonical imprinting. These observations suggest that H2Aub represses future bivalent genes during early embryogenesis without H3K27me3, but it is not required for the maintenance of non-canonical imprinting, which is mediated by maternal H3K27me3. Thus, our study reveals the distinct depositions and independent functions of H2Aub and H3K27me3 during early mammalian development.

Retinoic acid signaling is critical during the totipotency window in early mammalian development

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

Keep the fate: how chromatin regulators safeguard embryonic stem cell identity

Segregation of cells that form the embryo from those that produce the surrounding extra-embryonic tissues is critical for early mammalian development, but the regulatory layers governing these first cell fate decisions remain poorly understood. Recent work in The EMBO Journal identifies two chromatin regulators, Hdac3 and Dax1, that synergistically restrict the developmental potential of mouse embryonic stem cells and act as a lineage barrier to primitive endoderm formation.

Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

Most humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

Quantitative analysis of signaling responses during mouse primordial germ cell specification.

ABSTRACTDuring early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a−/−), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.

Cytoskeletal control of early mammalian development.

The cytoskeleton - comprising actin filaments, microtubules and intermediate filaments - serves instructive roles in regulating cell function and behaviour during development. However, a key challenge in cell and developmental biology is to dissect how these different structures function and interact in vivo to build complex tissues, with the ultimate aim to understand these processes in a mammalian organism. The preimplantation mouse embryo has emerged as a primary model system for tackling this challenge. Not only does the mouse embryo share many morphological similarities with the human embryo during its initial stages of life, it also permits the combination of genetic manipulations with live-imaging approaches to study cytoskeletal dynamics directly within an intact embryonic system. These advantages have led to the discovery of novel cytoskeletal structures and mechanisms controlling lineage specification, cell-cell communication and the establishment of the first forms of tissue architecture during development. Here we highlight the diverse organization and functions of each of the three cytoskeletal filaments during the key events that shape the early mammalian embryo, and discuss how they work together to perform key developmental tasks, including cell fate specification and morphogenesis of the blastocyst. Collectively, these findings are unveiling a new picture of how cells in the early embryo dynamically remodel their cytoskeleton with unique spatial and temporal precision to drive developmental processes in the rapidly changing in vivo environment.

Single-Cell Multiomic Approaches Reveal Diverse Labeling of the Nervous System by Common Cre-Drivers.

Neural crest development involves a series of dynamic, carefully coordinated events that result in human disease when not properly orchestrated. Cranial neural crest cells acquire unique multipotent developmental potential upon specification to generate a broad variety of cell types. Studies of early mammalian neural crest and nervous system development often use the Cre-loxP system to lineage trace and mark cells for further investigation. Here, we carefully profile the activity of two common neural crest Cre-drivers at the end of neurulation in mice. RNA sequencing of labeled cells at E9.5 reveals that Wnt1-Cre2 marks cells with neuronal characteristics consistent with neuroepithelial expression, whereas Sox10-Cre predominantly labels the migratory neural crest. We used single-cell mRNA and single-cell ATAC sequencing to profile the expression of Wnt1 and Sox10 and identify transcription factors that may regulate the expression of Wnt1-Cre2 in the neuroepithelium and Sox10-Cre in the migratory neural crest. Our data identify cellular heterogeneity during cranial neural crest development and identify specific populations labeled by two Cre-drivers in the developing nervous system.

Super-Enhancers and CTCF in Early Embryonic Cell Fate Decisions.

Cell fate decisions are the backbone of many developmental and disease processes. In early mammalian development, precise gene expression changes underly the rapid division of a single cell that leads to the embryo and are critically dependent on autonomous cell changes in gene expression. To understand how these lineage specifications events are mediated, scientists have had to look past protein coding genes to the cis regulatory elements (CREs), including enhancers and insulators, that modulate gene expression. One class of enhancers, termed super-enhancers, is highly active and cell-type specific, implying their critical role in modulating cell-type specific gene expression. Deletion or mutations within these CREs adversely affect gene expression and development and can cause disease. In this mini-review we discuss recent studies describing the potential roles of two CREs, enhancers and binding sites for CTCF, in early mammalian development.

A multiscale model via single-cell transcriptomics reveals robust patterning mechanisms during early mammalian embryo development.

During early mammalian embryo development, a small number of cells make robust fate decisions at particular spatial locations in a tight time window to form inner cell mass (ICM), and later epiblast (Epi) and primitive endoderm (PE). While recent single-cell transcriptomics data allows scrutinization of heterogeneity of individual cells, consistent spatial and temporal mechanisms the early embryo utilize to robustly form the Epi/PE layers from ICM remain elusive. Here we build a multiscale three-dimensional model for mammalian embryo to recapitulate the observed patterning process from zygote to late blastocyst. By integrating the spatiotemporal information reconstructed from multiple single-cell transcriptomic datasets, the data-informed modeling analysis suggests two major processes critical to the formation of Epi/PE layers: a selective cell-cell adhesion mechanism (via EphA4/EphrinB2) for fate-location coordination and a temporal attenuation mechanism of cell signaling (via Fgf). Spatial imaging data and distinct subsets of single-cell gene expression data are then used to validate the predictions. Together, our study provides a multiscale framework that incorporates single-cell gene expression datasets to analyze gene regulations, cell-cell communications, and physical interactions among cells in complex geometries at single-cell resolution, with direct application to late-stage development of embryogenesis.

Enhancement of Developmental Competence of Immature Oocytes Supplemented with Growth Factors in Culture Media

Background: In vitro embryo production is a valuable tool for understanding early mammalian development, therapeutic applications, excellent source for research in the field of developmental biology and production of valuable animals. The purpose of this study is to improve the production of in vitro cattle embryos using fibroblast and platelet derived growth factor as media supplement. Methods: Ovaries were collected from local abattoir in 0.9% saline (30-35°C) supplemented with antibiotics. Cumulus oocyte complexes were aspirated, washed 5-6 times and placed in maturation media supplemented with growth factors and cultured in 5% CO2 incubator at 38.5°C with maximum humidity. After 24 h oocytes were co-incubated with in vitro capacitated sperms for fertilization for 15-18 h and then presumptive zygotes were cultured for embryo development. Cleavage was observed after 40-42 h and embryos were co-cultured with oviductal cells for 7-9 days. Result: The highest cleavage and blastocyst formation rates were 55.93 ± 4.75, 57.06 ± 4.78, 51.24 ± 4.12 and 3.26 ±1.53, 2.42 ± 1.02, 2.70 ± 1.17 in FGF (1ng ml-1), PDGF (10 ng ml-1) and in combination of FGF and PDGF (1ng ml-1 each) respectively. It can be concluded that PDGF (10 ng ml-1) enhanced cleavage rate and FGF (1ng ml-1) enhanced blastocyst formation rate.

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

DNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells.

Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

The pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

Epithelial-Mesenchymal Transition Drives Three-Dimensional Morphogenesis in Mammalian Early Development.

From fertilization to onset of gastrulation, a mammalian embryo goes through several rounds of cellular morphogenesis resembling phenomena of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), collectively referred to as EMTs. How these EMT events play a role in shaping the three-dimensional (3-D) architecture of the developing embryo is not well-understood. In this review, we present a model in which cellular morphogenesis, represented primarily by dynamic changes in its epithelialization status, is the driving force of embryonic 3-D organization. This is achieved through the integration of three key components of mammalian early development, the pluripotency regulation, morphogenetic signaling, and biomechanical force anisotropy. Although cells in an early embryo do not exhibit full mesenchymal characteristics, our model underscores the importance of investigating molecular regulation of epithelial cell polarity and partial EMT/MET in understanding mammalian early development.

EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis.

Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.

Cell competition constitutes a barrier for interspecies chimerism.

Cell competition involves a conserved fitness-sensing process during which fitter cells eliminate neighbouring less-fit but viable cells1. Cell competition has been proposed as a surveillance mechanism to ensure normal development and tissue homeostasis, and has also been suggested to act as a barrier to interspecies chimerism2. However, cell competition has not been studied in an interspecies context during early development owing to the lack of an in vitro model. Here we developed an interspecies pluripotent stem cell (PSC) co-culture strategy and uncovered a previously unknown mode of cell competition between species. Interspecies competition between PSCs occurred in primed but not naive pluripotent cells, and between evolutionarily distant species. By comparative transcriptome analysis, we found that genes related to the NF-κB signalling pathway, among others, were upregulated in less-fit 'loser' human cells. Genetic inactivation of a core component (P65, also known as RELA) and an upstream regulator (MYD88)of the NF-κB complex in human cells could overcome the competition between human and mouse PSCs, thereby improving the survival and chimerism of human cells in early mouse embryos. These insights into cell competition pave the way for the study of evolutionarily conserved mechanisms that underlie competitive cell interactions during early mammalian development. Suppression of interspecies PSC competition may facilitate the generation of human tissues in animals.

DAXX Is a Crucial Factor for Proper Development of Mammalian Oocytes and Early Embryos.

The Death-domain associated protein 6 (DAXX) is an evolutionarily conserved and ubiquitously expressed multifunctional protein that is implicated in many cellular processes, including transcription, cellular proliferation, cell cycle regulation, Fas-induced apoptosis, and many other events. In the nucleus, DAXX interacts with transcription factors, epigenetic modifiers, and chromatin-remodeling proteins such as the transcription regulator ATRX—the α-thalassemia/mental retardation syndrome X-linked ATP-dependent helicase II. Accordingly, DAXX is considered one of the main players involved in chromatin silencing and one of the most important factors that maintain integrity of the genome. In this brief review, we summarize available data regarding the general and specific functions of DAXX in mammalian early development, with special emphasis on the function of DAXX as a chaperone of the histone variant H3.3. Since H3.3 plays a key role in the developmental processes, especially in the pronounced rearrangements of heterochromatin compartment during oogenesis and embryogenesis, DAXX can be considered as an important factor supporting proper development. Specifically, loss of DAXX affects the recruitment of ATRX, transcription of tandem repeats and telomere functions, which results in a decrease in the viability of early embryos.

Gastruloid Development Competence Discriminates Different States of Pluripotency.

SummaryFloating spheroidal aggregates of mouse embryonic stem cells can develop into polarized/elongated organoids, namely gastruloids. We set up a high-performing assay to measure gastruloid formation efficiency (GFE), and found that GFE decreases as pluripotency progresses from naive (GFE ≥ 95%) to primed (GFE = 0) state. Specifically, we show that primed EpiSCs fail to generate proper cell aggregates, while early-primed EpiLCs aggregate but eventually fail to develop into elongated gastruloids. Moreover, we characterized proline-induced cells (PiCs), a LIF-dependent reversible early-primed state of pluripotency, and show that PiCs are able to generate gastruloids (GFE ∼ 50%) and are also competent to differentiate into primordial germ cell-like cells. Thus, we propose the GFE assay as a valuable functional tool to discriminate different states of the pluripotency continuum.

Formation of a multi-layered 3-dimensional structure of the heterochromatin compartment during early mammalian development.

During embryogenesis in mammals, the 3-dimensional (3D) genome organization changes globally in parallel with transcription changes in a cell-type specific manner. This involves the progressive formation of heterochromatin, the best example of which is the inactive X chromosome (Xi) in females, originally discovered as a compact 3D structure at the nuclear periphery known as the Barr body. The heterochromatin formation on the autosomes and the Xi is tightly associated with the differentiation state and the developmental potential of cells, making it an ideal readout of the cellular epigenetic state. At a glance, the heterochromatin appears to be uniform. However, recent studies are beginning to reveal a more complex picture, with multiple hierarchical levels co-existing within the heterochromatin compartment. Such hierarchical levels appear to exist in the heterochromatin compartment on autosomes as well as on the Xi. Here, we review recent progress in our understanding of the 3D genome organization changes during the period of differentiation surrounding pluripotency in vivo and in vitro, with a focus on the heterochromatin compartment. We first look at the whole genome, then focus on the Xi, and discuss their differences and similarities. Finally, we present a unified view of how the heterochromatin compartment is formed and regulated during early development. In particular, we emphasize that there are multiple layers within the heterochromatic compartment on both the autosomes and the Xi, with regulatory mechanisms common and specific to each layer.

Directed Differentiation of Mouse Embryonic Stem Cells to Mesoderm, Endoderm, and Neuroectoderm Lineages.

The self-renewal and pluripotency features of mouse embryonic stem cells (mESCs) make them a great tool to study early mammalian development. Various signaling pathways that shape early mammalian development can be mimicked for in vitro mESC differentiation toward primitive lineages first and more specialized cell types later. Since the precise nature of the molecular mechanisms and the crosstalk between these signaling pathways is yet to be fully understood, there is a high level of variability in the efficiency and synchronicity among available differentiation protocols. Commitment of mESCs toward mesoderm, endoderm, or neuroectoderm lineages happens over only a few days and is highly efficient. Here, we provide protocols for the directed differentiation of mESCs toward these lineages in vitro.

Building the brain from scratch: Engineering region-specific brain organoids from human stem cells to study neural development and disease.

Human brain development is an intricate process that involves precisely timed coordination of cell proliferation, fate specification, neuronal differentiation, migration, and integration of diverse cell types. Understanding of these fundamental processes, however, has been largely constrained by limited access to fetal brain tissue and the inability to prospectively study neurodevelopment in humans at the molecular, cellular and system levels. Although non-human model organisms have provided important insights into mechanisms underlying brain development, these systems do not fully recapitulate many human-specific features that often relate to disease. To address these challenges, human brain organoids, self-assembled three-dimensional neural aggregates, have been engineered from human pluripotent stem cells to model the architecture and cellular diversity of the developing human brain. Recent advancements in neural induction and regional patterning using small molecules and growth factors have yielded protocols for generating brain organoids that recapitulate the structure and neuronal composition of distinct brain regions. Here, we first provide an overview of early mammalian brain development with an emphasis on molecular cues that guide region specification. We then focus on recent efforts in generating human brain organoids that model the development of specific brain regions and highlight endeavors to enhance the cellular complexity to better mimic the in vivo developing human brain. We also provide examples of how organoid models have enhanced our understanding of human neurological diseases and conclude by discussing limitations of brain organoids with our perspectives on future advancements to maximize their potential.