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Nature Feb 2023Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks. Here we use gene-regulatory networks inferred from...
Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms-mouse and human haematopoiesis, and zebrafish embryogenesis-and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.
Topics: Animals; Humans; Mice; Cell Differentiation; Embryonic Development; Gene Regulatory Networks; Phenotype; Transcription Factors; Zebrafish; Computer Simulation; Mesoderm; Hematopoiesis
PubMed: 36755098
DOI: 10.1038/s41586-022-05688-9 -
Biomedicine & Pharmacotherapy =... Oct 2019Endothelial-to-mesenchymal transition (EndMT) is closely related to the pathogenesis of various diseases, including cardiac fibrosis. Transforming growth factor...
Endothelial-to-mesenchymal transition (EndMT) is closely related to the pathogenesis of various diseases, including cardiac fibrosis. Transforming growth factor (TGF)-β1 strongly induces EndMT, and sirtuin 1 (SIRT1) may play vital roles in TGF-β/Smad pathway inhibition. This study aimed to determine whether SIRT1 activation inhibits EndMT, thereby attenuating cardiac fibrosis. Cardiac fibrosis was induced in C57BL/6 mice by subcutaneously injecting isoproterenol. SIRT1 was activated and then suppressed by intraperitoneally injecting resveratrol (RSV) and EX527, respectively. EndMT was induced by adding TGF-β1 to H5V cells and measured by immunofluorescence and western blot. The role of SIRT1 in EndMT was determined by lentivirus-mediated overexpression of SIRT1. Interactions between SIRT1 and Smad2/3 in the TGF-β/Smad2/3 pathway were examined by immunoprecipitation. SIRT1 activation upregulated CD31 and vascular endothelial-cadherin, and downregulated α-smooth muscle actin, fibroblast-specific protein 1, and vimentin. SIRT1 upregulated and EX527 inhibited TGF-β receptor 1 (TGF-βR1) and P-Smad2/3 expression, respectively. SIRT1 activation and overexpression by RSV/SRT2104 and lentivirus transfection, respectively, reduced TGF-β1-induced EndMT. SIRT1 and Smad2/3 interaction was shown by immunoprecipitation in vivo and in vitro. TGF-βR1 and P-Smad2/3 expression was downregulated and Smad2/3 nuclear translocation was inhibited. In conclusion, SIRT1 activated by RSV attenuated isoproterenol-induced cardiac fibrosis by regulating EndMT via the TGF-β/Smad2/3 pathway.
Topics: Animals; Cardiomegaly; Cell Line; Cell Nucleus; Collagen; Down-Regulation; Endothelium; Fibrosis; Isoproterenol; Male; Mesoderm; Mice, Inbred C57BL; Models, Biological; Myocardium; Phosphorylation; Protein Transport; Resveratrol; Sirtuin 1; Smad Proteins; Transforming Growth Factor beta
PubMed: 31351433
DOI: 10.1016/j.biopha.2019.109227 -
Cell Research Sep 2023Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well...
Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage development as well as underlying mechanisms remains obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling pathways synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast development and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights into human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.
Topics: Humans; Germ Layers; Embryo, Mammalian; Embryo Implantation; Endoderm; Mesoderm; Embryonic Development
PubMed: 37460804
DOI: 10.1038/s41422-023-00846-8 -
Nature Communications Aug 2022Cranial neural crest cells are an evolutionary innovation of vertebrates for craniofacial development and function, yet the mechanisms that govern the cell fate...
Cranial neural crest cells are an evolutionary innovation of vertebrates for craniofacial development and function, yet the mechanisms that govern the cell fate decisions of postmigratory cranial neural crest cells remain largely unknown. Using the mouse molar as a model, we perform single-cell transcriptome profiling to interrogate the cell fate diversification of postmigratory cranial neural crest cells. We reveal the landscape of transcriptional heterogeneity and define the specific cellular domains during the progression of cranial neural crest cell-derived dental lineage diversification, and find that each domain makes a specific contribution to distinct molar mesenchymal tissues. Furthermore, IGF signaling-mediated cell-cell interaction between the cellular domains highlights the pivotal role of autonomous regulation of the dental mesenchyme. Importantly, we reveal cell-type-specific gene regulatory networks in the dental mesenchyme and show that Foxp4 is indispensable for the differentiation of periodontal ligament. Our single-cell atlas provides comprehensive mechanistic insight into the cell fate diversification process of the cranial neural crest cell-derived odontogenic populations.
Topics: Animals; Cell Differentiation; Gene Expression Regulation, Developmental; Mesoderm; Mice; Morphogenesis; Neural Crest; Odontogenesis; Signal Transduction
PubMed: 35974052
DOI: 10.1038/s41467-022-32490-y -
Cell Feb 2023Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the...
Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.
Topics: Animals; Humans; Body Patterning; Embryonic Development; Gene Expression Regulation, Developmental; Mammals; Mesoderm; Morphogenesis; Somites; Wnt Signaling Pathway; Organoids
PubMed: 36657441
DOI: 10.1016/j.cell.2022.12.042 -
Nature Apr 2020The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts. Intestinal...
The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E (PGE). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE drives the expansion οf a population of Sca-1 reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1 cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1 cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE-Ptger4. Analyses of patient-derived organoids established that PGE-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE-Ptger4-Yap signalling axis.
Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, Ly; Arachidonic Acid; Carcinogenesis; Cell Cycle Proteins; Cell Proliferation; Colorectal Neoplasms; Cyclooxygenase 2; Dinoprostone; Female; Fibroblasts; Humans; Intestinal Mucosa; Intestines; Male; Membrane Proteins; Mesoderm; Mice; Neoplastic Stem Cells; Organoids; Paracrine Communication; Receptors, Prostaglandin E, EP4 Subtype; Single-Cell Analysis; Stem Cell Niche; YAP-Signaling Proteins
PubMed: 32322056
DOI: 10.1038/s41586-020-2166-3 -
Nature Communications Sep 2020Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of...
Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.
Topics: Adolescent; Adult; Animals; Cell Differentiation; Epithelial Cells; Female; Gene Expression Regulation, Developmental; Genetic Heterogeneity; Humans; Incisor; Male; Mesoderm; Mice; Mice, Inbred C57BL; Models, Animal; Molar; Odontoblasts; Stem Cells; Tooth; Young Adult
PubMed: 32968047
DOI: 10.1038/s41467-020-18512-7 -
Cell Feb 2023Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development...
Using four-dimensional whole-embryo light sheet imaging with improved and accessible computational tools, we longitudinally reconstruct early murine cardiac development at single-cell resolution. Nascent mesoderm progenitors form opposing density and motility gradients, converting the temporal birth sequence of gastrulation into a spatial anterolateral-to-posteromedial arrangement. Migrating precardiac mesoderm does not strictly preserve cellular neighbor relationships, and spatial patterns only become solidified as the cardiac crescent emerges. Progenitors undergo a mesenchymal-to-epithelial transition, with a first heart field (FHF) ridge apposing a motile juxta-cardiac field (JCF). Anchored along the ridge, the FHF epithelium rotates the JCF forward to form the initial heart tube, along with push-pull morphodynamics of the second heart field. In Mesp1 mutants that fail to make a cardiac crescent, mesoderm remains highly motile but directionally incoherent, resulting in density gradient inversion. Our practicable live embryo imaging approach defines spatial origins and behaviors of cardiac progenitors and identifies their unanticipated morphological transitions.
Topics: Mice; Animals; Heart; Cell Differentiation; Morphogenesis; Mesoderm; Embryo, Mammalian; Mammals
PubMed: 36736300
DOI: 10.1016/j.cell.2023.01.001 -
Nature Protocols Nov 2022Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent... (Review)
Review
Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a protocol to differentiate hPSCs into different types of organ-specific mesoderm, leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm, respectively, after 4-d cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7, the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60-90% pure liver septum transversum/mesothelium-like, 70-80% pure liver-like fibroblasts and populations of ~35% respiratory-like mesoderm, gastric-like mesoderm or esophageal-like mesoderm. This protocol can be performed by anyone with moderate experience differentiating hPSCs, provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.
Topics: Humans; Mice; Animals; Hedgehog Proteins; Mesoderm; Endoderm; Cell Differentiation; Pluripotent Stem Cells; Tretinoin; Lung
PubMed: 35978039
DOI: 10.1038/s41596-022-00733-3 -
Cell Feb 2024Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory...
Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.
Topics: Basic Helix-Loop-Helix Transcription Factors; Binding Sites; DNA; DNA-Binding Proteins; Gene Expression Regulation; Mesoderm; Transcription Factors; Humans; Animals; Mice; Extremities; Embryonic Development
PubMed: 38262408
DOI: 10.1016/j.cell.2023.12.032