Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the 45Ca-exchange method to determine free cellular Ca++, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca++). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca++] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca++ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca++] results in a release of the fusion-block and an increase in cellular Ca++ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca++] must exist. The latter suggests further that cell surface Ca++ was also critical.

A lactose-extractable lectin obtained from 14-16-d embryonic chick pectoral muscle and myotube muscle cultures by affinity chromatography inhibited myotube formation in culture . When applied to muscle cultures at 0 .09 Itg/ml, the purified lectin produced variable effects on the inhibition of myotube formation related to the time and length of application, suggesting that components of the culture medium and/or temperature produced inactivation . Hemagglutination assays showed that the lectin was inactivated by horse serum and by chick embryo extract but not by L-15 salt solution at 4°C . Incubation in L-15 solution at 37°C with or without 2 mM dithiothreitol resulted in inactivation in 2-3 h . To maximize the effect of the lectin on the inhibition of myotube formation, primary muscle cultures were grown in low [Ca"] medium to inhibit fusion, and then [Ca +2] was increased to elicit fusion in the absence and presence of lectin with solution renewal every 2 h. Without lectin, myotube formation was normal, whereas, with lectin, it was inhibited by 93% . Continued incubation at 37°C without renewal of lectin resulted in myotube formation, suggesting reversibility by lectin inactivation.It has been postulated that embryonic cells employ the appearance and/or disappearance of surface constituents during their development for specific tissue recognition (17,24,29) . Evidence for the appearance of such factors has been obtained for retinal cells and, more recently, for retinal cell membranes that show specificity for type, developmental stage, and absence of recognition factors in older embryos (8, 9) . Multivalent glycoproteins that promote species-specific sponge cell aggregation (10) and molecules that appear to be the receptors for these ligands on the cell surfaces (28) have been isolated in pure form . Simpson et al . (25) have characterized and purified a galactosebinding protein, discoidin, from the slime mold Dictyostelium discoideum in which the biosynthesis closely parallels the development of cell cohesiveness . Species-specificity is suggested because an-J . CELL BIOLOGY © The Rockefeller University Press -0021-9525/80/06/0617/09 $1 .00 Volume 85 June 1980 617-625 other slime mold, Polysphondylium pallidum, also contains a developmentally regulated cell surface lectin that reacts with species-specific cell surface oligosaccharides during cellular cohesion (26) .A /3-D-galactoside-specific lectin occurs in embryonic chick pectoral muscle and skeletal muscle cultures (4,7,19,27). The highest concentration appears between 8 and 16 d of development (19), which is also the period of highest myoblast fusion, suggesting a possible correlation between the lectin and the recognition-fusion process. The hemagglutinating activity of this lectin is inhibited by lactose and thiodigalactoside . Primary cultures of chicken (6) and rat skeletal (4) muscle are unaffected by thiodigalactoside ; however, the rat muscle cell lines L 6 (4, 7) and L 8 (4) are fusion inhibited .The objective of this study was to deter...

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Background: Embalmed cadavers in medical classes represent a potential source for collecting human tissues without the inherent problems of obtaining fresh or surgical specimens. Although the manner of fixation and vagaries of embalming techniques eliminate many such tissues for histological assessment, other techniques can be applied successfully to embalmed tissues. Pertinent to the present study, mast cells contain granules that are preserved under good fixation in formalde-hyde, a main ingredient in embalming fluids. Visualization of these granules is possible, even though the ultrastructure of these cells is not preserved. Methods: Two techniques for the visualization of connective tissue mast cells were compared using embalmed and fresh specimens: Alcian blue and avidin conjugated to fluorescein isothiocyanate (FITC-Avidin). Both will bind to mast cell granules, even in the presence of formaldehyde. Results: Although mast cell numbers in the connective tissue did not differ between embalmed and fresh tissues, comparisons between the techniques involved showed the FITC-Avidin technique to be possibly more sensitive, perhaps because of the increased contrast from the fluorescent dye. Conclusions: Thus for some studies, human cadavers may provide a valuable source of tissue. However, use of embalmed tissue necessitates ensuring good embalming, checking for dehydration, and proper storage until embedment. Anat.

Endogenous chicken muscle lectin isolated by lactose affinity chromatography inhibits myoblast fusion. Similar lectins isolated from embryonic brain, heart, and liver and from adult intestine exhibit the same ability. Elevated levels of any of these lectins canceled the inhibitory effect. Peanut agglutinin isolated by the same procedure had no effect at any concentration tested. Concanavalin A affected fusion only at high concentrations. Muscle lectin was shown to agglutinate myoblasts in microtiter plates, whereas exogenous addition in culture inhibited alignment as seen by time lapse microcinematography. Cell-to-cell communication between lectin-treated cells was shown by nucleotide exchange, and lectin-coated culture dishes did not affect cell attachment. Our evidence shows a lack of specificity to muscle, but suggests an aggregating capacity between cells, or possibly an interaction between the cell membrane and the extracellular matrix.