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Nucleic Acids Research Jun 2023Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite...
Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite functions in transcriptional activation and repression, but the mechanisms remain unclear. By converting mouse embryonic stem cells (ESCs) into neuroprogenitors, we defined how Tet1-/- cells switch from neuroectoderm fate to form mesoderm and endoderm. We identified the Wnt repressor Tcf7l1 as a TET1 target that suppresses Wnt/β-catenin and Nodal signalling. ESCs expressing catalytic dead TET1 retain neural potential but activate Nodal and subsequently Wnt/β-catenin pathways to generate also mesoderm and endoderm. At CpG-poor distal enhancers, TET1 maintains accessible chromatin at neuroectodermal loci independently of DNA demethylation. At CpG-rich promoters, DNA demethylation by TET1 affects the expression of bivalent genes. In ESCs, a non-catalytic TET1 cooperation with Polycomb represses primitive streak genes; post-lineage priming, the interaction becomes antagonistic at neuronal genes, when TET1's catalytic activity is further involved by repressing Wnt signalling. The convergence of repressive DNA and histone methylation does not inhibit neural induction in Tet1-deficient cells, but some DNA hypermethylated loci persist at genes with brain-specific functions. Our results reveal versatile switching of non-catalytic and catalytic TET1 activities based on genomic context, lineage and developmental stage.
Topics: Animals; Mice; 5-Methylcytosine; beta Catenin; DNA-Binding Proteins; Germ Layers; Genomics; Cell Differentiation; Proto-Oncogene Proteins
PubMed: 37021585
DOI: 10.1093/nar/gkad231 -
Frontiers in Reproductive Health 2023Discrete events and processes influence development of individual humans. Attribution of personhood to any individual human being cannot be disconnected from the... (Review)
Review
Discrete events and processes influence development of individual humans. Attribution of personhood to any individual human being cannot be disconnected from the underlying biological events and processes of early human development. Nonetheless, the philosophical, sociological and legal components that are integral to the meaning of the term as commonly used cannot be deduced from biology alone. The challenge for biomedical scientists to inform discussion in this arena then rests on profiling the key biological events and processes that must be assessed when considering how one might objectively reason about the task of superimposing the concept of personhood onto the developing biological entity of a potential human being. Endogenous genetic and epigenetic events and exogenous developmental processes diversify developmental trajectories of potential individual humans prior to livebirth. First, fertilization and epigenetic resetting of each individual's organismic clock to time zero ( = 0) at the gastrulation/primitive streak stage (day 15 of embryogenesis), are two discrete unseen biological events that impact a potential individual human's attributes. Second, those two discrete unseen biological events are immersed in the continuous developmental process spanning pre-fertilization and gestation, further driving individualization of diverse attributes of each future human before the third discrete and blatant biological event of parturition and livebirth. Exposures of the gravida to multiple diverse exogenous exposures means that morphogenesis and physiogenesis of every embryo/fetus has individualized attributes for its future human lifespan. Our proposed framework based on the biological discrete events and processes spanning pre-fertilization and prenatal development, implies that personhood should be incrementally attributed, and societal protections should be graduated and applied progressively across the pre-birth timespan.
PubMed: 37020713
DOI: 10.3389/frph.2023.1112935 -
Current Topics in Developmental Biology 2023Understanding how the body plan is established during embryogenesis remains a fundamental biological question. The Wnt/β-catenin signaling pathway plays a crucial and...
Understanding how the body plan is established during embryogenesis remains a fundamental biological question. The Wnt/β-catenin signaling pathway plays a crucial and highly conserved role in body plan formation, functioning to polarize the primary anterior-posterior (AP) or head-to-tail body axis in most metazoans. In this chapter, we focus on the roles that the mammalian Wnt/β-catenin pathway plays to prepare the pluripotent epiblast for gastrulation, and to elicit the emergence of multipotent axial progenitors from the caudal epiblast. Interactions between Wnt and retinoic acid (RA), another powerful family of developmental signaling molecules, in axial progenitors will also be discussed. Gastrulation movements and somitogenesis result in the anterior displacement of the RA source (the rostral somites and lateral plate mesoderm (LPM)), from the posterior Wnt source (the primitive streak (PS)), leading to the establishment of antiparallel gradients of RA and Wnt that control the self-renewal and successive differentiation of neck, trunk and tail progenitors.
Topics: Animals; Wnt Signaling Pathway; Body Patterning; Germ Layers; Mesoderm; Somites; Tretinoin; Cell Differentiation; Mammals
PubMed: 36967193
DOI: 10.1016/bs.ctdb.2023.01.010 -
ELife Mar 2023During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete,...
During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.
Topics: Animals; Gene Regulatory Networks; Nervous System; Chickens; Embryonic Development; Organizers, Embryonic; Vertebrates
PubMed: 36867045
DOI: 10.7554/eLife.73189 -
Methods in Molecular Biology (Clifton,... 2024The microfluidic amniotic sac embryoid (μPASE) is a human pluripotent stem cell (hPSC)-derived multicellular human embryo-like structure with molecular and...
The microfluidic amniotic sac embryoid (μPASE) is a human pluripotent stem cell (hPSC)-derived multicellular human embryo-like structure with molecular and morphological features resembling the progressive development of the early post-implantation human embryonic sac. The microfluidic device is specifically designed to control the formation of hPSC clusters and expose the clusters to different morphogen environments, allowing the development of μPASEs in a highly controllable, reproducible, and scalable fashion. The μPASE model displays human embryonic developmental landmarks such as lumenogenesis of the epiblast, amniotic cavity formation, and the specification of primordial germ cells and gastrulating cells (or mesendoderm cells). Here, we provide detailed instructions needed to reproduce μPASEs, including the immunofluorescence staining and cell retrieval protocols for characterizing μPASEs obtained under different experimental conditions.
Topics: Humans; Microfluidics; Germ Layers; Pluripotent Stem Cells; Embryo, Mammalian; Amnion; Cell Differentiation
PubMed: 36749485
DOI: 10.1007/7651_2022_470 -
Nature Communications Jan 2023The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization....
The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-β activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.
Topics: Animals; Humans; Transforming Growth Factor beta; Induced Pluripotent Stem Cells; Cell Differentiation; Pluripotent Stem Cells; Germ Layers; Mesoderm; Endoderm; Mammals
PubMed: 36681697
DOI: 10.1038/s41467-023-35965-8 -
Science Advances Jan 2023The morphology of gastrulation driving the internalization of the mesoderm and endoderm differs markedly among vertebrate species. It ranges from involution of...
The morphology of gastrulation driving the internalization of the mesoderm and endoderm differs markedly among vertebrate species. It ranges from involution of epithelial sheets of cells through a circular blastopore in amphibians to ingression of mesenchymal cells through a primitive streak in amniotes. By targeting signaling pathways controlling critical cell behaviors in the chick embryo, we generated crescent- and ring-shaped mesendoderm territories in which cells can or cannot ingress. These alterations subvert the formation of the chick primitive streak into the gastrulation modes seen in amphibians, reptiles, and teleost fish. Our experimental manipulations are supported by a theoretical framework linking cellular behaviors to self-organized multicellular flows outlined in detail in the accompanying paper. Together, this suggests that the evolution of gastrulation movements is largely determined by changes in a few critical cell behaviors in the mesendoderm territory across different species and controlled by a relatively small number of signaling pathways.
PubMed: 36598979
DOI: 10.1126/sciadv.abn5429 -
Advanced Science (Weinheim,... Feb 2023Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by...
Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes-associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY-box transcription factor 17 (SOX17) T/BRACHYURY cells. Molecular profiling and pathway analysis reveals a role for mechanotransduction-coupled wingless-type (WNT) signaling in orchestrating differentiation, which bears similarities to processes observed in whole organism models of development. After two days, the colonies form multilayered aggregates, which can be removed for further growth and differentiation. This approach demonstrates how materials alone can initiate gastrulation, thereby providing in vitro models of development and a tool to support organoid bioengineering efforts.
Topics: Humans; Epithelial-Mesenchymal Transition; Gastrulation; Germ Layers; Mechanotransduction, Cellular; Pluripotent Stem Cells; Cellular Microenvironment; YAP-Signaling Proteins; SOXF Transcription Factors
PubMed: 36519269
DOI: 10.1002/advs.202203614 -
Nature Dec 2022Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans,...
Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.
Topics: Animals; Humans; Mice; Gastrulation; Macaca fascicularis; Organogenesis; Single-Cell Analysis; Embryoid Bodies; Gene Expression Profiling; Primitive Streak; Neural Tube; Neural Crest; Hippo Signaling Pathway; Mesoderm; Stem Cells
PubMed: 36517595
DOI: 10.1038/s41586-022-05526-y -
Methods in Molecular Biology (Clifton,... 2024The development process of human embryo until blastocyst is well understood during the past 30 years, however, embryogenesis from blastocyst to pre-gastrulation was...
The development process of human embryo until blastocyst is well understood during the past 30 years, however, embryogenesis from blastocyst to pre-gastrulation was still remained a "black box". Limited by research materials and culture technologies, the "black box" is still unopened. We recently established an extended three-dimensional (3D) culture system of human blastocysts (Xiang et al., Nature 577(7791):537-542, 2020). The 3D embryo culture system could enable human blastocyst growing up to early primitive streak anlage stage in vitro. Here, we introduce the detail protocol and notes of culturing human 3D embryos.
Topics: Humans; Gastrulation; Embryo, Mammalian; Embryonic Development; Blastocyst; Embryo Culture Techniques
PubMed: 36515895
DOI: 10.1007/7651_2022_466