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Seminars in Cell & Developmental Biology May 2023Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures,... (Review)
Review
Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures, entirely in vitro. This recreation of embryonic form from naïve cells, known as synthetic morphogenesis, has important implications for both developmental biology and regenerative medicine. However, the capacity of stem cell-based models to recapitulate the morphogenetic cell behaviors that shape natural embryos remains unclear. In this review, we explore several examples of synthetic morphogenesis, with a focus on models of gastrulation and surrounding stages. By varying cell types, source species, and culture conditions, researchers have recreated aspects of primitive streak formation, emergence and elongation of the primary embryonic axis, neural tube closure, and more. Here, we describe cell behaviors within in vitro/ex vivo systems that mimic in vivo morphogenesis and highlight opportunities for more complete models of early development.
Topics: Gastrulation; Morphogenesis
PubMed: 35817656
DOI: 10.1016/j.semcdb.2022.07.002 -
Cells Jun 2022Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the...
Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin-fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin-fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin-integrin-ROCK-NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts.
Topics: Actomyosin; Animals; Biological Clocks; Chick Embryo; Fibronectins; Integrins; Somites
PubMed: 35805087
DOI: 10.3390/cells11132003 -
Metabolic Engineering Sep 2022CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9)...
CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.
Topics: CRISPR-Cas Systems; Gene Expression; Humans; Induced Pluripotent Stem Cells; Primitive Streak
PubMed: 35724832
DOI: 10.1016/j.ymben.2022.06.003 -
Development (Cambridge, England) Jun 2022Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the...
Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.
Topics: Animals; Blastoderm; Chick Embryo; Gastrula; Primitive Streak
PubMed: 35723262
DOI: 10.1242/dev.200303 -
Stem Cell Reports Jul 2022Embryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate,...
Embryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate, but aspects of their respective contributions remain unclear. Studying their impact on the spatial organization and developmental trajectories of micropatterned epiblast-like cell (EpiLC) colonies, we show that NODAL is required prior to BMP action to establish the mesoderm and endoderm lineages. The presence of BMP then forces NODAL and WNT to support the formation of posterior primitive streak (PS) derivatives, while its absence allows them to promote that of anterior PS derivatives. Also, a Nodal mutation elicits more severe patterning defects in vitro than in the embryo, suggesting that ligands of extra-embryonic origin can rescue them. These results support the implication of a combinatorial process in PS patterning and illustrate how the study of micropatterned EpiLC colonies can complement that of embryos.
Topics: Animals; Body Patterning; Endoderm; Gastrula; Germ Layers; Mesoderm; Mice; Primitive Streak; Transforming Growth Factor beta
PubMed: 35714597
DOI: 10.1016/j.stemcr.2022.05.009 -
Nature Sep 2022Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling...
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development and to advance stem-cell-based regenerative approaches. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
Topics: Animals; Callithrix; Cell Differentiation; Embryo, Mammalian; Endoderm; Female; Gastrulation; Gene Expression Profiling; Germ Layers; Humans; Pluripotent Stem Cells; Uterus
PubMed: 35709828
DOI: 10.1038/s41586-022-04953-1 -
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 -
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