ABSTRACT
Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.
Subject(s)
Calcitonin , Cell Lineage , Ciona intestinalis , Endoderm , Neural Crest , Neuroendocrine Cells , Animals , Endoderm/metabolism , Endoderm/cytology , Calcitonin/metabolism , Neuroendocrine Cells/metabolism , Neuroendocrine Cells/cytology , Ciona intestinalis/metabolism , Ciona intestinalis/embryology , Neural Crest/metabolism , Neural Crest/cytology , Chick Embryo , Mice , Vertebrates/embryology , Vertebrates/metabolism , Zebrafish/embryology , Lancelets/embryology , Lancelets/metabolism , Lancelets/genetics , Ultimobranchial Body/metabolismABSTRACT
Organoids, three-dimensional, stem cell-based structures that mimic the cellular and functional architecture of tissues, have emerged as an innovative in vitro tool. They offer highly efficient models for studying both embryonic development and disease progression processes. Colon organoids can also be generated from biopsies obtained during a colonoscopy. However, the invasive nature of biopsy collection poses practical challenges and introduces biases when studying patients who are already afflicted. Therefore, the use of iPSC-derived colon organoids can be considered a more practical approach for researchers and patients alike. Numerous protocols have been published for generating colon organoids from iPSCs. While most of these protocols share a common developmental process, some are labor-intensive or require additional equipment. Taking these considerations into account, we present a cost-effective and straightforward yet functionally robust colon organoid protocol: (1) definitive endoderm differentiation, (2) hindgut endoderm differentiation, and (3) maturation of colon spheroids into mature organoids.
Subject(s)
Cell Differentiation , Colon , Induced Pluripotent Stem Cells , Organoids , Organoids/cytology , Colon/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Cell Culture Techniques/methods , Endoderm/cytologyABSTRACT
Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.
Subject(s)
Actomyosin , Angiomotins , Cell Differentiation , Cell Size , Endoderm , Signal Transduction , Transcription Factors , YAP-Signaling Proteins , Humans , Endoderm/cytology , Endoderm/metabolism , Actomyosin/metabolism , YAP-Signaling Proteins/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Osmotic Pressure , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Cell Nucleus/metabolismABSTRACT
BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.
Subject(s)
Cell Differentiation , Endoderm , Epigenesis, Genetic , Human Embryonic Stem Cells , Humans , Endoderm/cytology , Endoderm/metabolism , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Pharynx/cytology , Pharynx/metabolism , Tretinoin/pharmacology , Tretinoin/metabolism , Gene Expression Regulation, Developmental , Transcription Factors/metabolism , Transcription Factors/genetics , MiceABSTRACT
Dysfunction of pancreatic δ cells contributes to the etiology of diabetes. Despite their important role, human δ cells are scarce, limiting physiological studies and drug discovery targeting δ cells. To date, no directed δ-cell differentiation method has been established. Here, we demonstrate that fibroblast growth factor (FGF) 7 promotes pancreatic endoderm/progenitor differentiation, whereas FGF2 biases cells towards the pancreatic δ-cell lineage via FGF receptor 1. We develop a differentiation method to generate δ cells from human stem cells by combining FGF2 with FGF7, which synergistically directs pancreatic lineage differentiation and modulates the expression of transcription factors and SST activators during endoderm/endocrine precursor induction. These δ cells display mature RNA profiles and fine secretory granules, secrete somatostatin in response to various stimuli, and suppress insulin secretion from in vitro co-cultured ß cells and mouse ß cells upon transplantation. The generation of human pancreatic δ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation studies in diabetes.
Subject(s)
Cell Differentiation , Pluripotent Stem Cells , Humans , Animals , Mice , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factor 2/pharmacology , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/cytology , Somatostatin-Secreting Cells/metabolism , Somatostatin-Secreting Cells/cytology , Endoderm/cytology , Endoderm/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 1/genetics , Pancreas/cytology , Pancreas/metabolism , Somatostatin/metabolism , Cell Lineage , Insulin/metabolism , Insulin SecretionABSTRACT
Cell fate decisions remain poorly understood at the molecular level. Embryogenesis provides a unique opportunity to analyze molecular details associated with cell fate decisions. Works based on model organisms have provided a conceptual framework of genes that specify cell fate control, for example, transcription factors (TFs) controlling processes from pluripotency to immunity1. How TFs specify cell fate remains poorly understood. Here we report that SALL4 relies on NuRD (nucleosome-remodeling and deacetylase complex) to interpret BMP4 signal and decide cell fate in a well-controlled in vitro system. While NuRD complex cooperates with SALL4 to convert mouse embryonic fibroblasts or MEFs to pluripotency, BMP4 diverts the same process to an alternative fate, PrE (primitive endoderm). Mechanistically, BMP4 signals the dissociation of SALL4 from NuRD physically to establish a gene regulatory network for PrE. Our results provide a conceptual framework to explore the rich landscapes of cell fate choices intrinsic to development in higher organisms involving morphogen-TF-chromatin modifier pathways.
Subject(s)
Bone Morphogenetic Protein 4 , Cell Differentiation , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Transcription Factors , Animals , Mice , Transcription Factors/metabolism , Transcription Factors/genetics , Bone Morphogenetic Protein 4/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Chromatin/metabolism , Gene Regulatory Networks , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Endoderm/metabolism , Endoderm/cytology , Signal Transduction , Cell Lineage , DNA-Binding ProteinsABSTRACT
Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.
Subject(s)
Apoptosis , Cell Lineage , DNA Methylation , Endoderm , Gene Expression Regulation, Developmental , Animals , Endoderm/cytology , Endoderm/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Phagocytosis , Mice, Inbred C57BL , Mice , Cell Differentiation , Female , Embryonic Development , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Mice, Transgenic , Gastrointestinal Tract/cytology , Gastrointestinal Tract/embryology , Gastrointestinal Tract/metabolismABSTRACT
Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies.
Subject(s)
Cell Differentiation , Single-Cell Analysis , Single-Cell Analysis/methods , Humans , Endoderm/cytology , Endoderm/metabolism , Hematopoiesis , Cell Lineage , Sequence Analysis, RNA/methods , Organoids/metabolism , Organoids/cytologyABSTRACT
Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor ß1 (TGF-ß1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-ß1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-ß1-treated cells refractory to Wnt signaling. Subsequently, TGF-ß1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-ß1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic ß cell yield for cell-based therapeutic applications.
Subject(s)
Bone Morphogenetic Proteins , Cell Differentiation , Endoderm , Human Embryonic Stem Cells , Wnt Signaling Pathway , Humans , Endoderm/cytology , Endoderm/metabolism , Cell Differentiation/drug effects , Wnt Signaling Pathway/drug effects , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Bone Morphogenetic Proteins/metabolism , Cell Lineage/drug effects , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Cell Line , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/pharmacologyABSTRACT
Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.
Subject(s)
Embryo, Mammalian , Endoderm , Gastrulation , Gene Expression Regulation, Developmental , Single-Cell Analysis , Animals , Endoderm/cytology , Endoderm/metabolism , Endoderm/embryology , Swine , Mice , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Cell Differentiation , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Transcriptome , Hepatocyte Nuclear Factor 3-beta/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , Cell Lineage , T-Box Domain Proteins/metabolism , T-Box Domain Proteins/genetics , Epithelial-Mesenchymal Transition/geneticsABSTRACT
The anterior-posterior axis of the mammalian embryo is laid down by the anterior visceral endoderm (AVE), an extraembryonic signaling center that is specified within the visceral endoderm. Current models posit that AVE differentiation is promoted globally by epiblast-derived Nodal signals, and spatially restricted by a BMP gradient established by the extraembryonic ectoderm. Here, we report spatially restricted AVE differentiation in bilayered embryo-like aggregates made from mouse embryonic stem cells that lack an extraembryonic ectoderm. Notably, clusters of AVE cells also form in pure visceral endoderm cultures upon activation of Nodal signaling, indicating that tissue-intrinsic factors can restrict AVE differentiation. We identify ß-catenin activity as a tissue-intrinsic factor that antagonizes AVE-inducing Nodal signals. Together, our results show how an AVE-like population can arise through interactions between epiblast and visceral endoderm alone. This mechanism may be a flexible solution for axis patterning in a wide range of embryo geometries, and provide robustness to axis patterning when coupled with signal gradients.
Subject(s)
Body Patterning , Cell Differentiation , Endoderm , Nodal Protein , Signal Transduction , beta Catenin , Animals , Endoderm/cytology , Endoderm/metabolism , Endoderm/embryology , beta Catenin/metabolism , Mice , Nodal Protein/metabolism , Nodal Protein/genetics , Germ Layers/metabolism , Germ Layers/cytology , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Embryo, Mammalian/cytologyABSTRACT
Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not consider cell type-specific variation across donors. Modelling this cell type-specific inter-individual variation could help elucidate cell type-specific biology and inform genes and cell types underlying complex traits. We therefore develop a new model to detect and quantify cell type-specific variation across individuals called CTMM (Cell Type-specific linear Mixed Model). We use extensive simulations to show that CTMM is powerful and unbiased in realistic settings. We also derive calibrated tests for cell type-specific interindividual variation, which is challenging given the modest sample sizes in scRNA-seq. We apply CTMM to scRNA-seq data from human induced pluripotent stem cells to characterize the transcriptomic variation across donors as cells differentiate into endoderm. We find that almost 100% of transcriptome-wide variability between donors is differentiation stage-specific. CTMM also identifies individual genes with statistically significant stage-specific variability across samples, including 85 genes that do not have significant stage-specific mean expression. Finally, we extend CTMM to partition interindividual covariance between stages, which recapitulates the overall differentiation trajectory. Overall, CTMM is a powerful tool to illuminate cell type-specific biology in scRNA-seq.
Subject(s)
Cell Differentiation , Induced Pluripotent Stem Cells , Sequence Analysis, RNA , Single-Cell Analysis , Transcriptome , Humans , Single-Cell Analysis/methods , Sequence Analysis, RNA/methods , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Cell Differentiation/genetics , Gene Expression Profiling/methods , RNA-Seq/methods , Endoderm/cytology , Endoderm/metabolismABSTRACT
Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.
Subject(s)
Blastocyst , Cell Differentiation , Endoderm , Animals , Endoderm/metabolism , Endoderm/cytology , Mice , Blastocyst/metabolism , Blastocyst/cytology , Cell Lineage , Octamer Transcription Factor-3/metabolism , Octamer Transcription Factor-3/genetics , Signal Transduction , Embryonic Development , Janus Kinases/metabolism , Gene Expression Regulation, Developmental , STAT Transcription Factors/metabolism , Transcription Factors/metabolism , Female , Embryo, Mammalian/metabolism , Embryo, Mammalian/cytologyABSTRACT
Optimizing the efficiency of definitive endoderm (DE) differentiation is necessary for the generation of diverse organ-like structures. In this study, we used the small molecule inhibitor saracatinib (SAR) to enhance DE differentiation of human embryonic stem cells and induced pluripotent stem cells. SAR significantly improved DE differentiation efficiency at low concentrations. The interaction between SAR and Focal Adhesion Kinase (FAK) was explored through RNA-seq and molecular docking simulations, which further supported the inhibition of DE differentiation by p-FAK overexpression in SAR-treated cells. In addition, we found that SAR inhibited the nuclear translocation of Yes-associated protein (YAP), a downstream effector of FAK, which promoted DE differentiation. Moreover, the addition of SAR enabled a significant reduction in activin A (AA) from 50 to 10 ng/mL without compromising DE differentiation efficiency. For induction of the pancreatic lineage, 10 ng/ml AA combined with SAR at the DE differentiation stage yielded a comparative number of PDX1+/NKX6.1+ pancreatic progenitor cells to those obtained by 50 ng/ml AA treatment. Our study highlights SAR as a potential modulator that facilitates the cost-effective generation of DE cells and provides insight into the orchestration of cell fate determination.
Subject(s)
Benzodioxoles , Cell Differentiation , Endoderm , Quinazolines , Signal Transduction , Humans , Cell Differentiation/drug effects , Endoderm/drug effects , Endoderm/cytology , Endoderm/metabolism , Benzodioxoles/pharmacology , Signal Transduction/drug effects , Quinazolines/pharmacology , Transcription Factors/metabolism , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Adaptor Proteins, Signal Transducing/metabolism , YAP-Signaling Proteins/metabolism , Focal Adhesion Kinase 1/metabolism , Focal Adhesion Kinase 1/genetics , Human Embryonic Stem Cells/drug effects , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Activins/metabolism , Molecular Docking SimulationABSTRACT
Nanoparticles have unique properties that make them useful in biomedicine. However, their extensive use raises concerns about potential hazards to the body. Therefore, it is crucial to establish effective and robust toxicology models to evaluate the developmental and functional toxicity of nanoparticles on the body. This article discusses the use of stem cells to study the developmental and functional toxicity of organs of endodermal origin due to nanoparticles. The study discovered that various types of nanoparticles have varying effects on stem cells. The application of stem cell models can provide a possibility for studying the effects of nanoparticles on organ development and function, as they can more accurately reflect the toxic mechanisms of different types of nanoparticles. However, stem cell toxicology systems currently cannot fully reflect the effects of nanoparticles on entire organs. Therefore, the establishment of organoid models and other advanced assessment models is expected to address this issue.
Subject(s)
Endoderm , Nanoparticles , Stem Cells , Animals , Nanoparticles/toxicity , Humans , Stem Cells/drug effects , Endoderm/drug effects , Endoderm/cytologyABSTRACT
BACKGROUND: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. METHODS: The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. RESULTS: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. CONCLUSIONS: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.
Subject(s)
Cell Differentiation , Endoderm , Wnt Signaling Pathway , Zebrafish , Humans , Wnt Signaling Pathway/genetics , Cell Differentiation/genetics , Endoderm/metabolism , Endoderm/cytology , Animals , Zebrafish/genetics , HEK293 Cells , Transcription Factor 7-Like 2 Protein/metabolism , Transcription Factor 7-Like 2 Protein/genetics , HCT116 Cells , Paired Box Transcription Factors/metabolism , Paired Box Transcription Factors/geneticsABSTRACT
The lineage decision that generates the epiblast and primitive endoderm from the inner cell mass (ICM) is a paradigm for cell fate specification. Recent mathematics has formalized Waddington's landscape metaphor and proven that lineage decisions in detailed gene network models must conform to a small list of low-dimensional stereotypic changes called bifurcations. The most plausible bifurcation for the ICM is the so-called heteroclinic flip that we define and elaborate here. Our re-analysis of recent data suggests that there is sufficient cell movement in the ICM so the FGF signal, which drives the lineage decision, can be treated as spatially uniform. We thus extend the bifurcation model for a single cell to the entire ICM by means of a self-consistently defined time-dependent FGF signal. This model is consistent with available data and we propose additional dynamic experiments to test it further. This demonstrates that simplified, quantitative and intuitively transparent descriptions are possible when attention is shifted from specific genes to lineages. The flip bifurcation is a very plausible model for any situation where the embryo needs control over the relative proportions of two fates by a morphogen feedback.
Subject(s)
Blastocyst , Cell Differentiation , Cell Lineage , Models, Biological , Animals , Mice , Blastocyst/metabolism , Blastocyst/cytology , Signal Transduction , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , Endoderm/cytology , Endoderm/metabolism , Germ Layers/cytology , Germ Layers/metabolismABSTRACT
Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell-derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement and external force, such as the spread out of cells from the primitive streak area, mimicked the homogeneous discoidal germ layer formation. These results were consistent with the development of amniotes, which suggests the effectiveness of human pluripotent stem cells as a good model for studying human embryogenesis.
Subject(s)
Cell Differentiation , Cell Movement , Endoderm , Mesoderm , Pluripotent Stem Cells , Humans , Endoderm/cytology , Mesoderm/cytology , Pluripotent Stem Cells/cytology , Computer SimulationABSTRACT
Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells (hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein (YAP) and phosphorylated YAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.
Subject(s)
Cell Differentiation , Cell Lineage , Endoderm , Human Embryonic Stem Cells , Humans , Cell Differentiation/physiology , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Endoderm/cytology , Endoderm/metabolism , YAP-Signaling Proteins/metabolism , Transcription Factors/metabolismABSTRACT
TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.