Changes in the surface coat of mesenchymal cells of mouse limb buds after enzymatic cell separation

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 145-155
Author(s):  
Evamaria Kohnert-Stavenhagen ◽  
Bernd Zimmermann
Keyword(s):  
Surface Layer ◽  
Cell Separation ◽  
Mesenchymal Cells ◽  
Ruthenium Red ◽  
Mouse Embryos ◽  
Surface Coat ◽  
Limb Buds ◽  
Partial Separation ◽  
Collagenase Treatment ◽  
Mouse Limb

Isolation of cells is nowadays performed by enzymatic means. The influence of such enzymes on the surface coat of mesenchymal and blastemal cells during the dissociation of limb buds from 11 -day-old mouse embryos was studied electron microscopically after staining with ruthenium red. EGTA or collagenase failed to bring about cell separation. The surface coat seemed to be unchanged after collagenase treatment. After EGTA an increase in extracellular filaments was observed. The proteases α-chymotrypsin, dispase II, papain, pronase P and trypsin (0·2%, 37 °C, 20 min) succeeded in completely dissociating limb buds. Apart from single granules, there was a detachment of the surface coat from the cells in all cases studied. Hyaluronidase led to only partial separation, but the detachment of the surface coat was almost complete, indicating a GAG-rich surface layer on these cells.

Ultrastructural aspects of early development of the fore-limb buds in the chick and the mouse

1965 ◽  
Vol 162 (988) ◽  
pp. 387-405 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Early Development ◽  
Secretory Granules ◽  
Free Cell ◽  
Mouse Embryos ◽  
Limb Bud ◽  
Chick Embryos ◽  
Limb Buds ◽  
Mouse Limb ◽  
A Chain

Light microscope investigations of the early development of the fore-limb buds in chick and mouse were made to guide electron microscope studies with these tissues. At the time of maximal development of the ectodermal apical ridge there is a higher concentration of cytoplasmic RNA in the apical ridge cells than in the other cells of the limb bud. Ultrastructural investigations showed that, in the mesoblast cells at the earliest stages, profiles of endoplasmic reticulum are often found attached to the outer nuclear membrane. Some what later, discontinuities of nuclear envelope occur by which the content of the nucleus may communicate with the endoplasmic reticulum. In the cytoplasm of the mesoblast cells at these stages there were many granules similar in form and size to secretory granules of gland cells. Ribosomes are in the polysomal condition. At stages later than 20 in chick and in 11-day-old mouse embryos, the mesoblast shows the character of a syncytial tissue. Epiblast cells possess all the characters of an epithelium with well developed junctional complexes. The desmosomes form a chain consisting of units equipped with individual dense plaques, but connected by continuous bundles of fibres running parallel to the chain. The free cell membrane of the epiblastic cells, particularly at early stages, forms numerous microvilli and single cilia. In later stages during the form action of the ectodermal apical ridge, cilia have been found between the cells. This fact indicates that when the apical ridge is formed ectodermal cells migrate towards the margin of the limb bud. At these stages microvilli are also found between the apical ridge cells where they contribute to the cell-to-cell adhesion. Beginning at stage 22 in chick embryos and from the 12th day in mouse embryos there are in cells of the apical ridge numerous and extensive Golgi systems spread throughout the cytoplasm. Some what later there appear successively lysosomes, cytolysomes and extranuclear necrotic centres. All these organelles manifest acid phosphatase activity and are thoughtto initiate the involutive process in the apical ridge. Pycnosis and karyorrhexis appear as the last stage of cellular degeneration. Degenerating cells undergo phagocytosis by neighbouring epithelial cells. A basement membrane is present at all stages of development of the chick and mouse limb buds. It is an acellular continuous structure lining the internal (basal) surface of the epiblast, but in chick embryos it shows discontinuities immediately under the apical ectodermal ridge at the time of its maximum development.

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry

1975 ◽  
Vol 19 (3) ◽  
pp. 621-644
Author(s):  
D.M. Dwyer
Keyword(s):  
Fine Structure ◽  
Cell Surface ◽  
Alcian Blue ◽  
Chondroitin Sulphate ◽  
Surface Membrane ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Cell Agglutination ◽  
Trypanosoma Lewisi ◽  
Cationic Compounds

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.

Vitamin A alters the internal viscosity of fragments of limb-bud mesenchyme

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 325-339
Author(s):  
T. E. Kwasigroch ◽  
D. M. Kochhar
Keyword(s):  
Retinoic Acid ◽  
Vitamin A ◽  
Mesenchymal Cells ◽  
Limb Bud ◽  
Mouse Limb ◽  
Vitamin A Acid ◽  
Internal Viscosity ◽  
Fusion Experiments

Two techniques were used to examine the effect of vitamin A compounds (vitamin A acid = retinoic acid and vitamin A acetate) upon the relative strengths of adhesion among mouse limb-bud mesenchymal cells. Treatment with retinoic acid in vivo and with vitamin A acetate in vitro reduced the rate at which the fragments of mesenchyme rounded-up when cultured on a non-adhesive substratum, but these compounds did not alter the behavior of tissues tested in fragment-fusion experiments. These conflicting results indicate that the two tests measure different activities of cells and suggest that treatment with vitamin A alters the property(ies) of cells which regulate the internal viscosity of tissues.

Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly

Development ◽  
2000 ◽  
Vol 127 (18) ◽  
pp. 4011-4021 ◽  
Author(s):  
M. Tanaka ◽  
M.J. Cohn ◽  
P. Ashby ◽  
M. Davey ◽  
P. Martin ◽  
...  
Keyword(s):  
Body Plan ◽  
Mouse Embryos ◽  
Apical Ectodermal Ridge ◽  
Central Feature ◽  
Chick Embryos ◽  
Limb Buds ◽  
Naturally Occurring ◽  
Induction Signal

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.

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