scholarly journals Isolation, Culture, and Differentiation of Blastema Cells from the Regenerating Caudal Fin of Zebrafish

Fishes ◽  
2020 ◽  
Vol 5 (1) ◽  
pp. 6 ◽  
Author(s):  
Parameswaran Vijayakumar ◽  
M. Leonor Cancela ◽  
Vincent Laizé
Keyword(s):  
Cell Differentiation ◽  
Primary Cultures ◽  
Cell Types ◽  
Blastema Cell ◽  
Caudal Fin ◽  
Epimorphic Regeneration ◽  
All Trans Retinoic Acid ◽  
Undifferentiated Cells ◽  
Wound Epidermis ◽  
Cost Efficient

The caudal fin of teleost fish has become an excellent system for investigating the mechanisms of epimorphic regeneration. Upon amputation of the caudal fin, a mass of undifferentiated cells, called blastema, proliferate beneath the wound-epidermis and differentiate into various cell types to faithfully restore the missing fin structures. Here we describe a protocol that can be used to isolate and culture blastema cells from zebrafish. Primary cultures were initiated from 36 h post-amputation (hpa) blastema and optimal cell growth was achieved using L-15 medium supplemented with 5% fetal bovine serum in plates either coated with fibronectin or uncoated. After seeding, zebrafish blastema cells formed a uniform culture and exhibited polygonal shapes with prominent nucleus, while various cell types were also observed after few days in culture indicating cell differentiation. Upon treatment with all-trans retinoic acid, zebrafish blastema cells differentiated into neuron-like and oligodendritic-like cells. Immunocytochemistry data also revealed the presence of mesenchymal and neuronal cells. The availability of blastema cell cultures could contribute to a better understanding of epimorphic regeneration by providing a mean to investigate the mechanisms underlying blastema cell differentiation. Furthermore, this protocol is simple, rapid, and cost-efficient, and can be virtually applied to the development of any fish blastema cell culture.

scholarly journals JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

2021 ◽  
Author(s):  
Manuel Tavares ◽  
Garima Khandelwal ◽  
Joanne Mutter ◽  
Keijo Viiri ◽  
Manuel Beltran ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Target Genes ◽  
Histone H3 ◽  
Endometrial Stromal Sarcoma ◽  
Cell Types ◽  
Polycomb Repressive Complex 2 ◽  
Stromal Sarcoma ◽  
N Terminus ◽  
Undifferentiated Cells

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain repression of genes specific for other cell types and is essential for cell differentiation. In endometrial stromal sarcoma, the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. Here, we show that JAZF1-SUZ12 dysregulates PRC2 composition, recruitment, histone modification, gene expression and cell differentiation. The loss of the SUZ12 N-terminus in the fusion protein disrupted interaction with the PRC2 accessory factors JARID2, EPOP and PALI1 and prevented recruitment of PRC2 from RNA to chromatin. In undifferentiated cells, JAZF1-SUZ12 occupied PRC2 target genes but gained a JAZF1-like binding profile during cell differentiation. JAZF1-SUZ12 reduced H3K27me3 and increased H4Kac at PRC2 target genes, and this was associated with disruption in gene expression and cell differentiation programs. These results reveal the defects in chromatin regulation caused by JAZF1-SUZ12, which may underlie its role in oncogenesis.

scholarly journals The Fine Structure of Blastema Cells and Differentiating Cartilage Cells in Regenerating Limbs of Amblystoma Larvae

1958 ◽  
Vol 4 (5) ◽  
pp. 583-592 ◽  
Author(s):  
Elizabeth D. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Fine Structure ◽  
Cell Types ◽  
Ground Substance ◽  
High Concentration ◽  
Blastema Cell ◽  
Undifferentiated Cells ◽  
Abundant Cytoplasm ◽  
Cartilage Cells ◽  
Cell Changes

Regenerating forelimbs of larval salamanders, Amblystoma punctatum, were fixed in OsO4 at various intervals after amputation and were sectioned for study with the electron microscope. The dedifferentiated cells comprising the early blastema were found to have a fine structure similar to that of other undifferentiated cells and to have lost all of the identifying morphological features of their tissues of origin. The cytoplasm of such cells is characterized by numerous free ribonucleoprotein granules and a discontinuous vesicular endoplasmic reticulum. The cells have more abundant cytoplasm and are in closer contact with each other than was previously realized. The layer of condensed ground substance investing most differentiated cell types is lacking. After a period of rapid cell division, the morphology of the blastema cell changes. Cytoplasm is now sparse and contains a high concentration of free ribonucleoprotein granules, but little endoplasmic reticulum. The differentiating cartilage cell, however, develops an extensive, highly organized endoplasmic reticulum and the Golgi apparatus also appears to become more highly differentiated and more extensive at this time. Small vesicles appear throughout the cytoplasm at the time the new cisternae originate and may contribute to their formation. These and other changes in the cytoplasmic organelles are discussed.

The Murine Misty Mutation: Phenotypic Effects on Melanocytes, Platelets and Brown Fat

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 381-390
Author(s):  
Elena V Sviderskaya ◽  
Edward K Novak ◽  
Richard T Swank ◽  
Dorothy C Bennett
Keyword(s):  
Coat Color ◽  
Adenine Nucleotide ◽  
Primary Cultures ◽  
Cell Types ◽  
Nucleotide Metabolism ◽  
Brown Fat ◽  
Melanocyte Stimulating Hormone ◽  
Prolonged Bleeding Time ◽  
Adenine Nucleotide Metabolism

Abstract Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

Interpolation of microtubules into cortical arrays during cell elongation and differentiation in roots of Azolla pinnata

1979 ◽  
Vol 37 (1) ◽  
pp. 411-442
Author(s):  
A.R. Hardham ◽  
B.E. Gunning
Keyword(s):  
Cell Differentiation ◽  
Cell Walls ◽  
Unit Length ◽  
Cell Types ◽  
High Rate ◽  
Wall Thickening ◽  
Cortical Microtubules ◽  
Azolla Pinnata ◽  
Outer Cortex ◽  
Cortical Cells

Longitudinal sections of roots of Azolla pinnata R. Br. were prepared for electron microscopy so that cortical microtubules could be counted along the longitudinal walls in cell files in the endodermis, pericycle, and inner and outer cortex, and in sieve and xylem elements. With the exception of the xylem, where there are no transverse cell divisions, each file of cells commences with its initial cell and then possesses a zone of concomitant cell expansion and transverse cell division, followed, after completion of the divisions, by a zone of terminal cell differentiation. The cells augment their population of cortical microtubules as they elongate and divide, showing a net increase of up to 0.6 micron of polymerized microtubule length per min. Two main sub-processes were found: (i) When a longitudinal wall is first formed it is supplied with a higher number of microtubules per unit length of wall than it will have later, when it is being expanded. This initial quota becomes diluted as the second sub-process commences. (ii) The cells interpolate new microtubules at a rate which is characteristic of the cell, and, in the endodermis, of the face of the cell, while the cell elongates. Most cell types thus maintain a set density of cortical microtubules while they elongate and divide. Comparisons of endodermal cells in untreated controls, and roots that had been treated with colchicine, low temperature, or high pressure indicate that the initial quota of microtubules, and the later interpolations, and differentially sensitive to microtuble perturbations. Three types of behaviour, all related to changes in the cell walls, were noted as cortex, xylem and sieve element cells entered their respective phases of cell differentiation. The cortical cells expanded in all dimensions, and the interpolation of microtubules diminished or ceased. The sieve elements continued to elongate, and interpolated at a high rate, reaching unusually high densities of microtubules when the cell walls were being thickened. During this period a net increase of 2.0 micron of polymerized microtubule length per min was calculated. Thereafter interpolation ceased and the density of microtubules declined. The sample applied to developing xylem except that, because wall-thickening is localized rather than widespread, the rise and subsequent fall in the density of microtubules was less marked. The data are discussed in relation to the participation of microtubules in wall deposition and to the hypothesis that cortical microtubules arise in discrete zones along the edges of cells.

Heparin stimulates the proliferation of intestinal epithelial cells in primary culture

1994 ◽  
Vol 107 (2) ◽  
pp. 401-411
Author(s):  
N. Flint ◽  
F.L. Cove ◽  
G.S. Evans
Keyword(s):  
Growth Factors ◽  
Heparan Sulphate ◽  
Primary Cultures ◽  
Cell Types ◽  
Heparin Binding ◽  
Epithelial Proliferation ◽  
Inhibition Of Proliferation ◽  
Trophic Effect ◽  
Heparin Binding Growth Factors

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

scholarly journals Enhancement of All-trans Retinoic Acid-Induced HL-60 Cell Differentiation by Thalidomide and Its Metabolites

2005 ◽  
Vol 28 (3) ◽  
pp. 563-564 ◽  
Author(s):  
Tomomi Noguchi ◽  
Chihiro Shinji ◽  
Hisayoshi Kobayashi ◽  
Makoto Makishima ◽  
Hiroyuki Miyachi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

scholarly journals Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble β-Glucan

2021 ◽  
Vol 12 ◽  
Author(s):  
Dimitar B. Iliev ◽  
Guro Strandskog ◽  
Mehrdad Sobhkhez ◽  
Jack A. Bruun ◽  
Jorunn B. Jørgensen
Keyword(s):  
Immune System ◽  
Atlantic Salmon ◽  
Primary Cultures ◽  
Head Kidney ◽  
Cell Types ◽  
Mononuclear Phagocytes ◽  
Immunostimulatory Activity ◽  
Complement Receptor 3 ◽  
Bacteria And Fungi

β‐Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

scholarly journals Cellular heterogeneity and lineage restriction during mouse digit tip regeneration at single cell resolution

2019 ◽  
Author(s):  
Gemma L. Johnson ◽  
Erick J. Masias ◽  
Jessica A. Lehoczky
Keyword(s):  
Single Cell ◽  
Limb Regeneration ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Specific Gene ◽  
Genetic Lineage ◽  
Epimorphic Regeneration ◽  
Related Cell ◽  
Lower Vertebrates ◽  
Single Cell Rna Sequencing

ABSTRACTInnate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses in mice, the digit tip blastema has been defined as a population of heterogeneous, lineage restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restriction of closely related cell types. In this report we present single cell RNA sequencing of over 38,000 cells from mouse digit tip blastemas and unamputated control digit tips and generate an atlas of the cell types participating in digit tip regeneration. We define the differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals an early blastema fibroblast population expressing a novel regeneration-specific gene, Mest.

scholarly journals Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation

1995 ◽  
Vol 309 (3) ◽  
pp. 905-912 ◽  
Author(s):  
J W Kok ◽  
T Babia ◽  
K Klappe ◽  
D Hoekstra
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Differentiation ◽  
Plasma Membranes ◽  
Cell Types ◽  
Short Chain ◽  
Synthetic Activity ◽  
Intact Cells ◽  
Ht29 Cells ◽  
Extensive Degradation

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

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