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Cell Stem Cell Jun 2022In this issue of Cell Stem Cell, Simunovic et al. (2022) establish embryoids by combining embryonic and extraembryonic components derived from human pluripotent stem...
In this issue of Cell Stem Cell, Simunovic et al. (2022) establish embryoids by combining embryonic and extraembryonic components derived from human pluripotent stem cells. The embryoids resemble human embryos cultured to post-implantation stages in vitro with regard to morphology, symmetry breaking, and the formation of primitive streak-like cell types.
Topics: Embryo Implantation; Embryo, Mammalian; Humans; Pluripotent Stem Cells
PubMed: 35659870
DOI: 10.1016/j.stem.2022.05.009 -
Methods in Molecular Biology (Clifton,... 2022During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules...
During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. Progress on these issues is slowed by the difficulty to record or perturb the patterning events in real time, especially in mammalian embryos that develop in utero. Because they permit easy observation and manipulation, in vitro model systems offer an exciting opportunity to dissect the rules governing the organization of the mammalian gastrula. For instance, it is sufficient to cultivate human embryonic stem cells on micropatterned substrates to reveal their self-organization potential. We present here a method to obtain micropatterned mouse Epiblast Like Cells colonies, providing a convenient way to compare spatial organization of mouse and human pluripotent stem cells and to complement the characterization of mutant embryos in a controlled environment.
Topics: Animals; Cell Differentiation; Embryo, Mammalian; Gastrula; Human Embryonic Stem Cells; Humans; Mammals; Pluripotent Stem Cells
PubMed: 35486251
DOI: 10.1007/978-1-0716-2281-0_18 -
Communications Biology Apr 2022Previously, we have shown that the translocation of Grainyhead-like 3 (GRHL3) transcription factor from the nucleus to the cytoplasm triggers the switch from canonical...
Previously, we have shown that the translocation of Grainyhead-like 3 (GRHL3) transcription factor from the nucleus to the cytoplasm triggers the switch from canonical Wnt signaling for epidermal differentiation to non-canonical Wnt signaling for epithelial morphogenesis. However, the molecular mechanism that underlies the cytoplasmic localization of GRHL3 protein and that activates non-canonical Wnt signaling is not known. Here, we show that ubiquitin-specific protease 39 (USP39), a deubiquitinating enzyme, is involved in the subcellular localization of GRHL3 as a potential GRHL3-interacting protein and is necessary for epithelial morphogenesis to up-regulate expression of planar cell polarity (PCP) components. Notably, mouse Usp39-deficient embryos display early embryonic lethality due to a failure in primitive streak formation and apico-basal polarity in epiblast cells, resembling those of mutant embryos of the Prickle1 gene, a crucial PCP component. Current findings provide unique insights into how differentiation and morphogenesis are coordinated to construct three-dimensional complex structures via USP39.
Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Differentiation; Cell Polarity; DNA-Binding Proteins; LIM Domain Proteins; Mammals; Mice; Morphogenesis; Transcription Factors; Up-Regulation
PubMed: 35440748
DOI: 10.1038/s42003-022-03254-7 -
Development (Cambridge, England) May 2022In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret...
In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the 'neighbourhood watch' model) illuminates the classic 'French Flag Problem' and how positional information is interpreted by a sheet of cells in a large developing system.
Topics: Animals; Chick Embryo; Gastrula; Gastrulation; Germ Layers
PubMed: 35438131
DOI: 10.1242/dev.200295 -
Nucleic Acids Research Jul 2022The standard analysis pipeline for single-cell RNA-seq data consists of sequential steps initiated by clustering the cells. An innate limitation of this pipeline is that...
The standard analysis pipeline for single-cell RNA-seq data consists of sequential steps initiated by clustering the cells. An innate limitation of this pipeline is that an imperfect clustering result can irreversibly affect the succeeding steps. For example, there can be cell types not well distinguished by clustering because they largely share the global structure, such as the anterior primitive streak and mid primitive streak cells. If one searches differentially expressed genes (DEGs) solely based on clustering, marker genes for distinguishing these types will be missed. Moreover, clustering depends on many parameters and can often be subjective to manual decisions. To overcome these limitations, we propose MarcoPolo, a method that identifies informative DEGs independently of prior clustering. MarcoPolo sorts out genes by evaluating if the distributions are bimodal, if similar expression patterns are observed in other genes, and if the expressing cells are proximal in a low-dimensional space. Using real datasets with FACS-purified cell labels, we demonstrate that MarcoPolo recovers marker genes better than competing methods. Notably, MarcoPolo finds key genes that can distinguish cell types that are not distinguishable by the standard clustering. MarcoPolo is built in a convenient software package that provides analysis results in an HTML file.
Topics: Algorithms; Biomarkers; Cluster Analysis; Gene Expression Profiling; RNA-Seq; Sequence Analysis, RNA; Single-Cell Analysis; Software; Exome Sequencing
PubMed: 35420135
DOI: 10.1093/nar/gkac216 -
Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids.ELife Apr 2022Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging...
Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.
Topics: Animals; Cadherins; Cell Differentiation; Cell Movement; Endoderm; Gastrulation; Germ Layers; Mammals; Mice
PubMed: 35404233
DOI: 10.7554/eLife.59371 -
Developmental Biology May 2022T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal...
T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T, which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1, known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors.
Topics: Animals; Ectopic Gene Expression; Embryonic Development; Gene Expression Regulation, Developmental; Mesoderm; Mice; Somites; T-Box Domain Proteins
PubMed: 35276131
DOI: 10.1016/j.ydbio.2022.02.010 -
Nature Communications Feb 2022During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and...
During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.
Topics: Actin Cytoskeleton; Animals; Apoptosis Regulatory Proteins; Caco-2 Cells; Cell Polarity; Dogs; Embryo Culture Techniques; Embryo, Mammalian; Epithelium; Female; Gastrulation; Germ Layers; Humans; Madin Darby Canine Kidney Cells; Mice; Mice, Transgenic; Morphogenesis; Mutation; Primitive Streak; Receptors, Neuropeptide Y; Stress, Mechanical; Tight Junctions; Tumor Suppressor Proteins
PubMed: 35177595
DOI: 10.1038/s41467-022-28590-4 -
Development (Cambridge, England) Mar 2022Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later...
Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations.
Topics: Animals; Birds; Brain; Cell Movement; Ectoderm; Gastrula; Germ Layers
PubMed: 35132990
DOI: 10.1242/dev.199999 -
Genesis (New York, N.Y. : 2000) Feb 2022Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFβ pathway. In vitro methodologies...
Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFβ pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFβ family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFβ pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFβ/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFβ/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.
Topics: Activins; Cell Differentiation; Endoderm; Germ Layers; Nodal Protein; Stem Cells; Transforming Growth Factor beta
PubMed: 35104045
DOI: 10.1002/dvg.23466