Circus movements in dissociated cells in normal and hybrid frog embryos

1981 ◽  
Vol 49 (1) ◽  
pp. 205-216
Author(s):  
K.E. Johnson ◽  
M.R. Adelman
Keyword(s):  
Progressive Increase ◽  
Normal Embryo ◽  
Gastrula Stage ◽  
Isolated Cells ◽  
Embryo Cells ◽  
Dividing Cells ◽  
Dissociated Cells ◽  
Small Clusters ◽  
Early Gastrula

Circus movements, involving circumferential rotation of a hyaline cytoplasmic blister and endoplasmic flow, occur in EDTA-dissociated gastrula stage Rana pipiens embryos. Such cell movements occur in very few cells taken from pre-gastrula stage embryos. During gastrulation, there is a progressive increase in the proportion of a population of cells that is engaged in circus movements. Circus movements do not occur in dividing cells. Individual cells in culture, as well as small clusters of cells in vitro, are jostled about in an apparently aimless fashion over short distances by circus movements, although the translocation of masses of cells over long distances is substantially greater than the translocation of isolated cells. In an early gastrula stage normal embryo, cells from around the site of blastopore invagination are most active in circus movements. Cells taken from different stages of arrested hybrid embryos show variable depression in the formation of rotating hyaline blebs. Aggregates of cells from arrested hybrid embryos are also relatively immobile in culture. The morphogenetic significance of circus movements in normal embryos and gastrula-arrest hybrid embryos is discussed.

Expression of N-CAM precedes neural induction in Pleurodeles waltl (urodele, amphibian)

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 675-683 ◽  
Author(s):  
J.P. Saint-Jeannet ◽  
F. Foulquier ◽  
C. Goridis ◽  
A.M. Duprat
Keyword(s):  
Monoclonal Antibody ◽  
Nervous System ◽  
Xenopus Laevis ◽  
Neural Induction ◽  
Gastrula Stage ◽  
Pleurodeles Waltl ◽  
Early Gastrula

The appearance and localization of N-CAM during neural induction were studied in Pleurodeles waltl embryos and compared with recent contradictory results reported in Xenopus laevis. A monoclonal antibody raised against mouse N-CAM was used. In the nervous system of Pleurodeles, it recognized two glycoproteins of 180 and 140×10(3) M(r) which are the Pleurodeles equivalent of N-CAM-180 and -140. Using this probe for immunohistochemistry and immunocytochemistry, we showed that N-CAM was already expressed in presumptive ectoderm at the early gastrula stage. In late gastrula embryos, a slight increase in staining was observed in the neurectoderm, whereas the labelling persisted in the noninduced ectoderm. When induced ectodermal cells were isolated at the late gastrula stage and cultured in vitro up to 14 days, a faint polarized labelling of cells was observed initially. During differentiation, the staining increased and became progressively restricted to differentiating neurons.

Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis

1976 ◽  
Vol 22 (3) ◽  
pp. 575-583
Author(s):  
K.E. Johnson
Keyword(s):  
Xenopus Laevis ◽  
Cell Contact ◽  
Embryonic Cells ◽  
Gastrula Stage ◽  
Isolated Cells ◽  
Daughter Cells ◽  
Cytoplasmic Protrusion ◽  
Endodermal Cells ◽  
In Cells

Circus movements, which involve the circumferential rotation of a hyaline cytoplasmic protrusion, occur in cells obtained by EDTA dissociation of gastrula-stage Xenopus laevis embryos. Only a few dissociated blastula-stage cells show circus movements, more early gastrula-stage cells show them, and nearly all late gastrula-stage cells show them. Circus movements cease in cells prior to mitosis and begin again in daughter cells after mitosis is completed. In early gastrulae, only 17% of prospective endodermal cells show circus movements while 79% of prospective mesodern, archenteric roof, and posterior neural ectoderm do so. Isolated cells as well as groups of cells in vitro are often propelled by circus movements. There is an obvious antagonism between cell contact and circus movements. The morphogenetic significance of circus movements and blebbing locomotion is discussed.

Embryonic development of Xenopus studied in a cell culture system with tissue-specific monoclonal antibodies

Development ◽  
1989 ◽  
Vol 105 (1) ◽  
pp. 53-59 ◽  
Author(s):  
S. Mitani ◽  
H. Okamoto
Keyword(s):  
Cellular Differentiation ◽  
Marginal Zone ◽  
Deep Layer ◽  
Epidermal Differentiation ◽  
Cell Culture System ◽  
Tissue Specific ◽  
A Cell ◽  
Dissociated Cells ◽  
Early Gastrula

An in vitro microculture system of early gastrula cells of Xenopus laevis has been developed; deep layer cells from the lateral marginal zone (prospective somite region) or ventral ectoderm (prospective epidermis region) were fully dissociated, and the desired number of each (1–100) was distributed into a microculture well and cultured under appropriate conditions. When examined with the tissue-specific Mabs (Mu1 for muscle and E3 for epidermis, respectively), a substantial portion of the deep layer cells from the two regions followed their respective normal embryonic fates. It was found that reproducible cellular differentiation was dependent on the intimate reaggregation of dissociated cells and on the size of the resultant aggregate. About 20 lateral marginal zone cells were found to be sufficient, when put into a culture well, for supporting successful muscle differentiation, whereas about 100 ventral ectoderm cells were necessary for epidermal differentiation.

Expression of tenascin in response to neural induction in amphibian embryos

Development ◽  
1988 ◽  
Vol 104 (3) ◽  
pp. 511-524 ◽  
Author(s):  
J.-F. Riou ◽  
D.-L. Shi ◽  
M. Chiquet ◽  
J.-C. Boucaut
Keyword(s):  
Cell Migration ◽  
Neural Crest Cell ◽  
Neural Induction ◽  
Gastrula Stage ◽  
Directed Cell Migration ◽  
Pleurodeles Waltl ◽  
Migration Pathways ◽  
Early Gastrula

The expression of tenascin, a constituent of extracellular matrix (ECM), was studied during the embryonic development of the amphibian Pleurodeles waltl. An antiserum to chick fibroblast tenascin was shown to cross-react with the homologous molecule of the amphibian. Immunostaining of embryo sections with anti-tenascin antiserum revealed that tenascin appears just after the completion of neurulation. At the tailbud stage, tenascin is present in the ECM located at sites of directed cell migration (neural crest cell migration pathways, extension of the pronephretic duct) and mesenchyme condensation (endocardium, aortic arches). The accumulation of tenascin immunoreactivity in the embryonic ECM is correlated with the synthesis of the 220×103Mr polypeptide of the molecule. To provide data on the patterning of tenascin, ectoderm and dorsal blastoporal lip isolated at early gastrula stage were cultured for a period of 3 days. Epidermal vesicles differentiating from isolated ectoderm completely lack tenascin. Conversely, axial mesoderm derivatives present in cultured dorsal blastoporal lip were found to produce tenascin. Neural induction of ectoderm isolated at early gastrula stage was performed in vitro with the dorsal blastoporal lip or concanavalin A. The induced neural tissue was found to accumulate tenascin. Spemann experiments confirmed in vivo that tenascin is expressed by ectodermal cells as a response to neural induction.

Initiation of mesodermal cell migration and spreading relative to gastrulation in the urodele amphibian Pleurodeles waltl

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 351-363 ◽  
Author(s):  
D.-L. Shi ◽  
T. Darribere ◽  
K.E. Johnson ◽  
J.-C. Boucaut
Keyword(s):  
Cell Migration ◽  
Marginal Zone ◽  
Gastrula Stage ◽  
Cell Stage ◽  
Animal Pole ◽  
Mesodermal Cell ◽  
Marginal Cells ◽  
Pleurodeles Waltl ◽  
Early Gastrula

We have investigated the autonomous migration of marginal cells and their interactions with extracellular matrix (ECM) located on the inner surface of the blastocoel roof in the urodele amphibian, Pleurodeles waltl, using a novel in vitro migration assay. Animal hemispheres containing equatorial cells removed at different cleavage stages and dorsal marginal zone (DMZ) explants of early gastrula stage were cultured either on fibronectin (FN)-coated or ECM-conditioned substrata. In explanted animal hemispheres, dorsal marginal cells showed autonomous migration on FN-coated substratum at the same time as the onset of gastrulation in control embryos. They acquired this capacity at least at the 32-cell stage, whereas lateral and ventral marginal cells acquired it after the 64-cell stage. DMZ outgrowths of early gastrula stage exhibited autonomous spreading on both substrata. In addition, we showed that they spread preferentially toward the animal pole when deposited on substratum conditioned by the dorsal roof of the blastocoel. By culturing dissociated marginal cells on ECM- conditioned substratum, we also found that increased spreading capacity of marginal cells was related to the initiation of their migration. A comparative study of the migration of marginal cells in ultraviolet (u.v.)-irradiated and normal embryos was also made. The results indicate that dorsal marginal cell migration was absent or dramatically reduced by u.v.-irradiation. These results suggest that the differential acquisition in the spreading capacity both in timing and in intensity around the marginal zone was correlated with the sequential involution of mesodermal cells in the course of gastrulation.

Application of Transmission Electron Microscope and Scanning Electron Microscope in experimental tumor studies

1990 ◽  
Vol 48 (3) ◽  
pp. 306-307
Author(s):  
Gao Fengming
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Malignant Transformation ◽  
Transmission Electron Microscope ◽  
Experimental Tumor ◽  
Transmission Electron ◽  
Embryo Cells ◽  
Tumor Studies ◽  
Scanning Electron

Transmission electron microscope(TEM) and scanning electron microscope(SEM) were widely used in experimental tumor studies. They are useful for evaluation of cellular transformation in vitro, classification of histological types of tumors and treating effect of tumors. We have obtained some results as follows:1. Studies on the malignant transformation of mammalian cells in vitro. Syrian golden hamster embryo cells(SGHEC) were transformed in vitro by ThO2 and/or ore dust. In a few days after dust added into medium, some dust crystals were phagocytized. Two weeks later, malignant transformation took place. These cells were of different size, nuclear pleomorphism, numerous ribosomes, increasing of microvilli on cell surface with various length and thickness, and blebs and ruffles(Figs. 1,2). Myelomonocytic leukemic transformation of mouse embryo cells(MEC) was induced in vitro by 3H-TdR. Transformed cells were become round from fusiform. The number of mitochondria and endoplasmic reticulum was reduced, ribosomes and nucleoli increased, shape of nuclei irregular, microvilli increased, and blebs and ruffles appeared(Fig. 3).

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽  
2021 ◽  
Author(s):  
Xiaoxiao Wang ◽  
Yunlong Xiang ◽  
Yang Yu ◽  
Ran Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Large Set ◽  
Mouse Escs ◽  
Epiblast Stem Cells ◽  
Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

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