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PLoS Genetics Oct 2022The evolutionarily conserved RNA helicase DDX6 is a central player in post-transcriptional regulation, but its role during embryogenesis remains elusive. We here show...
The evolutionarily conserved RNA helicase DDX6 is a central player in post-transcriptional regulation, but its role during embryogenesis remains elusive. We here show that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 knockout (KO) mouse embryos and employing an in vitro epiblast-like cell (EpiLC) induction system. Our study unveils that DDX6 is an important BMP signaling regulator. Deletion of Ddx6 causes the aberrant upregulation of the negative regulators of BMP signaling, which is accompanied by enhanced expression of Nodal and related genes. Ddx6 KO pluripotent cells acquire higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal and Eomes expression in the primitive streak likely promotes endoderm cell fate specification while inhibiting mesoderm differentiation. We also genetically dissected major DDX6 pathways by generating Dgcr8, Dcp2, and Eif4enif1 KO models in addition to Ddx6 KO. We found that the miRNA pathway mutant Dgcr8 KO phenocopies Ddx6 KO, indicating that DDX6 mostly works along with the miRNA pathway during early development, whereas its P-body-related functions are dispensable. Therefore, we conclude that DDX6 prevents aberrant upregulation of BMP signaling inhibitors by participating in miRNA-mediated gene silencing processes. Overall, this study delineates how DDX6 affects the development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.
Topics: Animals; Bone Morphogenetic Proteins; Cell Differentiation; DEAD-box RNA Helicases; Gastrulation; Gene Silencing; Mice; MicroRNAs; Proto-Oncogene Proteins; RNA-Binding Proteins; Transforming Growth Factor beta
PubMed: 36197846
DOI: 10.1371/journal.pgen.1009967 -
Stem Cell Reports Oct 2022The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the...
The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kβ) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kβ non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear β-catenin accumulation. β-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kβ epigenetic control of EZH2/β-catenin might be modulated to direct stem cell differentiation.
Topics: Cell Differentiation; Embryonic Stem Cells; Enhancer of Zeste Homolog 2 Protein; Gene Expression; Humans; JNK Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Primitive Streak; beta Catenin
PubMed: 36179694
DOI: 10.1016/j.stemcr.2022.09.003 -
Development Genes and Evolution Dec 2022During primitive streak formation in the chick embryo, cells undergo mesendoderm specification and convergent extension at the same time and in the same cells. Previous...
During primitive streak formation in the chick embryo, cells undergo mesendoderm specification and convergent extension at the same time and in the same cells. Previous work has implicated cVG1 (GDF3) as a key factor for induction of primitive streak identity and positioning the primitive streak, whereas FGF signalling was implicated in regulating cell intercalation via regulation of components of the WNT-planar cell polarity (PCP) pathway. FGF has also been reported to be able to induce a primitive streak (but lacking the most axial derivatives such as notochord/prechordal mesendoderm). These signals emanate from different cell populations in the embryo, so how do they interact to ensure that the same cells undergo both cell intercalation and acquire primitive streak identity? Here we begin to address this question by examining in more detail the ability of the two classes of signals in regulating the two developmental events. Using misexpression of inducers and/or exposure to inhibitors and in situ hybridisation, we study how these two signals regulate expression of Brachyury (TBXT) and PRICKLE1 as markers for the primitive streak and the PCP, respectively. We find that both signals can induce both properties, but while FGF seems to be required for induction of the streak by cVG1, it is not necessary for induction of PRICKLE1. The results are consistent with cVG1 being a common regulator for both primitive streak identity and the initiation of convergent extension that leads to streak elongation.
Topics: Animals; Chick Embryo; Primitive Streak; Gastrulation; Signal Transduction; Cell Polarity; Gastrula
PubMed: 36149507
DOI: 10.1007/s00427-022-00696-1 -
International Journal of Molecular... Sep 2022Progesterone treatment is commonly employed to promote and support pregnancy. While maternal tissues are the main progesterone targets in humans and mice, its receptor...
Progesterone treatment is commonly employed to promote and support pregnancy. While maternal tissues are the main progesterone targets in humans and mice, its receptor (PGR) is expressed in the murine embryo, questioning its function during embryonic development. Progesterone has been previously associated with murine blastocyst development. Whether it contributes to lineage specification is largely unknown. Gastrulation initiates lineage specification and generation of the progenitors contributing to all organs. Cells passing through the primitive streak (PS) will give rise to the mesoderm and endoderm. Cells emerging posteriorly will form the extraembryonic mesodermal tissues supporting embryonic growth. Cells arising anteriorly will contribute to the embryonic heart in two sets of distinct progenitors, first (FHF) and second heart field (SHF). We found that PGR is expressed in a posterior-anterior gradient in the PS of gastrulating embryos. We established in vitro differentiation systems inducing posterior (extraembryonic) and anterior (cardiac) mesoderm to unravel PGR function. We discovered that PGR specifically modulates extraembryonic and cardiac mesoderm. Overexpression experiments revealed that PGR safeguards cardiac differentiation, blocking premature SHF progenitor specification and sustaining the FHF progenitor pool. This role of PGR in heart development indicates that progesterone administration should be closely monitored in potential early-pregnancy patients undergoing infertility treatment.
Topics: Animals; Cell Differentiation; Female; Gastrula; Gastrulation; Humans; Mesoderm; Mice; Pregnancy; Progesterone; Receptors, Progesterone
PubMed: 36142249
DOI: 10.3390/ijms231810307 -
Translational Research in Anatomy Jun 2022The purpose of this study is to characterize a full-term conjoined twins' cadaver curated by Dr. Jacob Henle sometime between 1844 and 1852 and demonstrate digital...
BACKGROUND
The purpose of this study is to characterize a full-term conjoined twins' cadaver curated by Dr. Jacob Henle sometime between 1844 and 1852 and demonstrate digital distribution of an old and rare medical museum specimen using an extended reality (XR) model workflow.
METHODS
The cadaver (Preparation 296) is in the Department of Anatomy and Cell Biology at the University of Heidelberg. An XR display workflow comprises image capture, segmentation, and visualization using CT/MR scans derived from the cadaver. Online radiology presentation to medical students focuses on diagnostic characteristics of anatomical systems depicted with XR models.
RESULTS
Developmental defects in Preparation 296 include duplicated supradiaphragmatic structures and abnormal osteological features. Subdiaphragmatically, the gut is continuous on the right, but terminates at the distal esophagus on the left. One large liver occupies the abdomen with one spleen located on the left side. Observations suggest duplication of the primitive streak and separate notochords rostrally. Duplication occurs near the yolk sac and involves midgut formation while secondary midline fusion of the upper extremities and ribs likely results from the proximity of the embryos during development. Medical students access the model with device agnostic software during the curricular topic "Human Body Plan" that includes embryology concepts covering mechanisms of twinning.
CONCLUSIONS
The workflow enables ease-of-access XR visualizations of an old and rare museum specimen. This study also demonstrates digital distribution and utilization of XR models applicable to embryology education.
PubMed: 36133355
DOI: 10.1016/j.tria.2022.100171 -
Nature Communications Sep 2022Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains...
Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.
Topics: Animals; Cell Differentiation; Humans; Mesoderm; Mice; Ribosomal Proteins; Ribosomes; Stem Cells; Wnt Signaling Pathway
PubMed: 36123354
DOI: 10.1038/s41467-022-33263-3 -
Development (Cambridge, England) Oct 2022The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia...
The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development.
Topics: Animals; Cell Differentiation; Embryonic Stem Cells; Endoderm; Hypoxia; Mammals; Primitive Streak
PubMed: 36102628
DOI: 10.1242/dev.200679 -
Cell Stem Cell Sep 2022Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids)...
Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.
Topics: Animals; Blastocyst; Cell Lineage; Ectoderm; Embryo, Mammalian; Germ Layers; Humans; Single-Cell Analysis
PubMed: 36055194
DOI: 10.1016/j.stem.2022.08.009 -
International Journal of Molecular... Aug 2022The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular...
H NMR Metabolite Monitoring during the Differentiation of Human Induced Pluripotent Stem Cells Provides New Insights into the Molecular Events That Regulate Embryonic Chondrogenesis.
The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.
Topics: Cell Differentiation; Chondrogenesis; Epigenesis, Genetic; Humans; Induced Pluripotent Stem Cells; Proton Magnetic Resonance Spectroscopy
PubMed: 36012540
DOI: 10.3390/ijms23169266 -
Stem Cell Research & Therapy Jul 2022In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for...
BACKGROUND
In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared.
METHODS
We characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose.
RESULTS
Podocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability.
CONCLUSIONS
We found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.
Topics: Animals; Diabetes Mellitus; Diabetic Nephropathies; Glucose; Humans; Induced Pluripotent Stem Cells; Kidney Glomerulus; Mice; Podocytes
PubMed: 35883199
DOI: 10.1186/s13287-022-03040-6