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Proceedings of the National Academy of... Feb 2022In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural,...
In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.
Topics: Animals; Body Patterning; Chick Embryo; Endoderm; Gastrula; Mesoderm; Nervous System; Notochord; Organizers, Embryonic; Stem Cell Niche; Stem Cells
PubMed: 35101917
DOI: 10.1073/pnas.2108935119 -
Frontiers in Cell and Developmental... 2021MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic...
MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic development, including the cardiovascular system. In this study, we analyze the expression profiles of different microRNAs during early cardiac development. By using whole mount hybridization in developing chick embryos, with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a, miR-106a, and miR-100 expression during early stages of embryogenesis (HH3 to HH17). We also correlated those findings with putative microRNA target genes by means of mirWalk and TargetScan analyses. Our results demonstrate a dynamic expression pattern in cardiac precursor cells from the primitive streak to the cardiac looping stages for miR-23b, miR-130a, and miR-106a. Additionally, miR-100 is later detectable during cardiac looping stages (HH15-17). Interestingly, the /inflow tract was shown to be the most representative cardiac area for the convergent expression of the four microRNAs. Through analysis we revealed that distinct Hox family members are predicted to be targeted by the above microRNAs. We also identified expression of several Hox genes in the at stages HH11 and HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and explants, we demonstrated the modulation of the different Hox clusters, Hoxa, Hoxb, Hoxc, and Hoxd genes, by these microRNAs. Furthermore, we correlated the negative modulation of several Hox genes, such as Hoxa3, Hoxa4, Hoxa5, Hoxc6, or Hoxd4. Finally, we demonstrated through a dual luciferase assay that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a, supporting a possible role of these microRNAs in Hox gene modulation during differentiation and compartmentalization of the posterior structures of the developing venous pole of the heart.
PubMed: 35087828
DOI: 10.3389/fcell.2021.767954 -
Stem Cell Reports Feb 2022The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this...
The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we identified retinoic acid (RA) as a critical mediator of early neural induction triggered by TGFβ or Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification, up to the emergence of somite and neural fates. In the absence of the RA-synthesizing enzyme Aldh1a2, a sensitive RA reporter revealed a hitherto unidentified residual RA signaling that specified neural fate. Genetic evidence showed that the RA-degrading enzyme Cyp26a1 protected PS-like cells from neural induction, even in the absence of TGFβ and Wnt antagonists. Overall, we characterized a multi-layered control of RA levels that regulates early neural differentiation in an in vitro PS-like system.
Topics: Aldehyde Dehydrogenase 1 Family; Animals; Benzamides; Cell Differentiation; Dioxoles; Ectoderm; Mice; Mouse Embryonic Stem Cells; Neurons; Primitive Streak; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Signal Transduction; Tretinoin
PubMed: 35063128
DOI: 10.1016/j.stemcr.2021.12.014 -
PloS One 2022DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic...
DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.
Topics: 5-Methylcytosine; Animals; Cell Differentiation; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; DNA Methyltransferase 3A; DNA Modification Methylases; DNA-Binding Proteins; Genomic Imprinting; Heterochromatin; Mice; Mice, Knockout; Mouse Embryonic Stem Cells; Retroelements
PubMed: 34986190
DOI: 10.1371/journal.pone.0262277 -
Science (New York, N.Y.) Dec 2021The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian and avian embryos and helps confer anterior-posterior and dorsal-ventral... (Review)
Review
The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian and avian embryos and helps confer anterior-posterior and dorsal-ventral spatial information to early differentiating cells during gastrulation. Its recapitulation in vitro may facilitate derivation of tissues and organs with in vivo–like complexity. Proper understanding of the primitive streak and what it entails in human development is key to achieving such research objectives. Here we provide an overview of the primitive streak and conclude that this structure is neither conserved nor necessary for gastrulation or early lineage diversification. We offer a model in which the primitive streak is viewed as part of a morphologically diverse yet molecularly conserved process of spatial coordinate acquisition. We predict that recapitulation of the primitive streak is dispensable for development in vitro.
Topics: Animals; Biological Evolution; Embryo, Mammalian; Embryo, Nonmammalian; Gastrulation; Gene Expression Regulation, Developmental; Humans; Morphogenesis; Phylogeny; Primitive Streak; Vertebrates
PubMed: 34855481
DOI: 10.1126/science.abg1727 -
Developmental Dynamics : An Official... May 2022Defects in secondary neurulation play an important role in neural tube defects. Researchers have investigated the processes of secondary neurulation and caudal body...
BACKGROUND
Defects in secondary neurulation play an important role in neural tube defects. Researchers have investigated the processes of secondary neurulation and caudal body formation mainly by microscopic observations and molecular experiments. Although conventional histology is a powerful tool for observing the details of morphology, it has limitations in the presentation of gross three-dimensional (3D) configurations of small embryos. The goal of this study was to visualize secondary neurulation and related structures in chick embryos in Hamburger and Hamilton (HH) stages 10-22 using microCT.
RESULTS
The gross morphology of the chick embryo of various developmental stages was well visualized using microCT. Also, the detailed structures of the caudal cell mass (CCM) were presented starting from HH stage 12 to stage 16. The spatiotemporal relationship of CCM with the floor plate of the neural tube and notochord was shown. The dynamic changes of the chordoneural hinge, the cavitation of the secondary neural tube, and the primitive streak were described throughout the early stages of secondary neurulation.
CONCLUSIONS
By utilizing the advantages of the microCT technique, our study shed light on the secondary neurulation in early-stage chick embryos and this can be the 3D reference for related structures.
Topics: Animals; Chick Embryo; Imaging, Three-Dimensional; Neural Tube; Neurulation; Notochord; X-Ray Microtomography
PubMed: 34811830
DOI: 10.1002/dvdy.441 -
Journal of Microbiology & Biology... Dec 2021In classroom studies of mammalian embryology, students must fully grasp the cellular and tissue remodeling needed to initiate gastrulation to ensure comprehension of...
Utilizing Playdoh in the Classroom to Construct a 3D Model Depicting Cellular Movements and Tissue Remodeling during Human Gastrulation, Early Organogenesis, and Embryonic Folding.
In classroom studies of mammalian embryology, students must fully grasp the cellular and tissue remodeling needed to initiate gastrulation to ensure comprehension of forthcoming developmental processes such as tissue specification and organogenesis. However, quickly and completely communicating three-dimensional concepts such as gastrulation, neurulation, and embryonic folding through common two-dimensional tools such as PowerPoint is challenging for students because this method lacks the spatial orientation needed to fully understand development. Therefore, professors can utilize active learning approaches with 3D-modeling clay to aid students in visualizing developmental changes. 3D-modeling of the developmental processes focused on cell and tissue movements for the initiation of gastrulation and organogenesis is limited in published literature/videos. Therefore, this activity fills in the modeling gap by focusing on the detachment and movement of the epiblast cell through the primitive streak to generate the three germ layers, neural tube folding, cardiogenesis, and the anatomical position of the early brain and heart to drive embryonic folding. The usage of this hands-on learning tool will assist lecturers in preventing early gaps in knowledge while students first construct the model and allows for correction in misunderstandings by utilizing the complete model in discussions after construction.
PubMed: 34804320
DOI: 10.1128/jmbe.00123-21 -
Cell Discovery Oct 2021Paternal life experiences impact offspring health via germline, and epigenetic inheritance provides a potential mechanism. However, global reprogramming during offspring...
Sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress via evading offspring embryonic reprogramming.
Paternal life experiences impact offspring health via germline, and epigenetic inheritance provides a potential mechanism. However, global reprogramming during offspring embryogenesis and gametogenesis represents the largest hurdle to conceptualize it. Yet, detailed characterization of how sperm epigenetic alterations carrying "environmental memory" can evade offspring embryonic reprogramming remains elusive. Here, mice exposed to long-term restraint stress were employed to study the mechanisms underlying inter- and transgenerational effects of paternal exposure to a long-term psychological stress. We found that stress could induce paternal inheritance of reproductive, behavioral, and metabolic disorders. Bisulfite methylation profiling of 18 sperm and 12 embryo samples of three consecutive generations identified inter- and transgenerational inheritance of paternal Differential DNA Methylation Regions (DMRs) at frequencies ~11.36% and 0.48%, respectively. These DMRs related to genes with functional implications for psychological stress response, and tissue inheritance of these DMRs passed paternal disorders epigenetically to offspring. More importantly, these DMRs evaded offspring embryonic reprogramming through erasure and subsequent reestablishment, but not via un-erasure way. Nonetheless, their reestablishment proportions in the primitive streak (E7.5) stage were altered. Furthermore, sncRNA-seq revealed that stress-induced tsRNA, miRNA and rsRNA dysregulation in paternal sperm might play important roles in DMRs occurrence and paternal inheritance. These finding implied that sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress, and highlighted the possible underlying molecular mechanism.
PubMed: 34711814
DOI: 10.1038/s41421-021-00343-5 -
Stem Cell Reviews and Reports Jan 2022Mesenchymal Stem Cells (MSCs), as an adult stem cell type, are used to treat various disorders in clinics. However, derivation of homogenous and adequate amount of MSCs...
Mesenchymal Stem Cells (MSCs), as an adult stem cell type, are used to treat various disorders in clinics. However, derivation of homogenous and adequate amount of MSCs limits the regenerative treatment potential. Although mesoderm is the main source of mesenchymal progenitors during embryonic development, neuromesodermal progenitors (NMPs), reside in the primitive streak during development, is known to differentiate into paraxial mesoderm. In the current study, we generated NMPs from human embryonic stem cells (hESC), subsequently derived MSCs and characterized this cell population in vitro and in vivo. Using a bFGF and CHIR induced NMP formation protocol followed by serum containing culture conditions; here we show that MSCs can be generated from NMPs identified by not only the expression of T/Bra and Sox 2 but also FLK-1/PDGFRα in our study. NMP-derived MSCs were plastic adherent fibroblast like cells with colony forming capacity and trilineage (osteo-, chondro- and adipo-genic) differentiation potential. In the present study, we demonstrate that NMP-derived MSCs have an endothelial tendency which might be related to their FLK-1+/PDGFRα + NMP origin. NMP-derived MSCs displayed a protein expression profile of characterized MSCs. Growth factor and angiogenesis related pathway proteins were similarly expressed in NMP-derived MSCs and characterized MSCs. NMP-derived MSCs keep characteristics after short-term and long-term freeze-thaw cycles and localized into bone marrow followed by tail vein injection into NOD/SCID mice. Together, these data showed that hESC-derived NMPs might be used as a precursor cell population for MSC derivation and could be used for in vitro and in vivo research.
Topics: Animals; Female; Humans; Mesenchymal Stem Cells; Mesoderm; Mice; Mice, Inbred NOD; Mice, SCID; Pregnancy; Receptor, Platelet-Derived Growth Factor alpha
PubMed: 34669151
DOI: 10.1007/s12015-021-10281-0 -
Molecular Biology of the Cell Dec 2021Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We...
Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B that requires sequences in the NOT box domain of CNOT3 and regulates MAPK/ERK signaling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse embryonic stem cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4, and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis.
Topics: A549 Cells; Animals; Aurora Kinase B; Cell Differentiation; Cell Survival; Cells, Cultured; Endoderm; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Mesoderm; Mice; Mouse Embryonic Stem Cells; Mutation; Phosphorylation; Transcription Factors
PubMed: 34613789
DOI: 10.1091/mbc.E21-02-0089