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R F Foster

Publications and source records attributed to R F Foster.

14 recordsLinked to original sources
PubMed1993

In vitro and in vivo expression of alpha 7 integrin and desmin define the primary and secondary myogenic lineages.

Skeletal muscle fibers form during two periods of development and differ biochemically, functionally and in their morphology. Primary fibers develop in the rat hindlimb during Days 14 to 16 of embryogenesis. These fibers are subsequently surrounded by secondary fibers that eventually constitute the bulk of muscle mass in the limbs. We have used the expression of the alpha 7 muscle laminin binding integrin (Song et al., J. Cell Biol. 117, 643-657, 1992) and the intermediate filament protein desmin to identify myogenic cells at distinct stages of development both in vitro and in vivo. The phenotypes of these cells, determined by immunofluorescence microscopy, discriminate two lineages and indicate that the development of primary and secondary muscle fibers is regulated by multiple mechanisms. The cells which compose the primary myogenic lineage are derived from a population of precursor cells that is in part present in the Day 12 embryo limb bud and which do not express either alpha 7 integrin or desmin. These precursor cells develop into cells that express desmin, but not alpha 7, and which subsequently mature into replicating myoblasts that are competent to undergo terminal differentiation. This maturation process requires the in vivo environment of the Day 13 embryo limb. The alpha 7 integrin and slow myosin heavy chain are first expressed in primary muscle cells well after the onset of terminal differentiation. Some cells that give rise to secondary muscle fibers also are present in the Day 12 embryo hindlimb. The precursors of secondary fibers will develop into cells which express either alpha 7 integrin or desmin and subsequently into replicating myoblasts that express both proteins. Upon terminal differentiation of secondary myoblasts there is an increase in the expression of both alpha 7 integrin and desmin. The temporal regulation of expression of these proteins indicates that the environment of the limb plays a role in the maturation of precursors of both lineages. At least two roles of alpha 7 integrin during myogenesis are related to its association with beta 1 integrin and its function as a laminin receptor. Laminin selectively maintains the proliferation of secondary myoblasts and modulates their shape and mobility in vitro. This responsiveness of secondary myoblasts to laminin corresponds to the time when laminin is a major component of the extracellular matrix, when there is an expansion of the population of secondary myoblasts, and when the alpha 7 integrin is expressed on secondary myoblasts in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis.

H36 is a 120,000-D membrane glycoprotein that is expressed during the differentiation of skeletal muscle. H36 cDNA clones were isolated from a lambda UniZapXR rat myotube cDNA library and sequenced. The deduced amino acid sequence demonstrates that H36 is a novel integrin alpha chain that shares extensive homology with other alpha integrins that includes: (a) the GFFKR sequence found in all alpha integrins; (b) a single membrane spanning region; (c) conservation of 18 of 22 cysteines; and (d) a protease cleavage site found in the non-I region integrin alpha chains. The cytoplasmic domain of H36 is unique and additional regions of nonhomology further indicate H36 is distinct from all other alpha chains. In keeping with current nomenclature we designate this alpha chain alpha 7. Northern blots demonstrate that expression of H36-alpha 7 mRNA is regulated both early in the development of the myogenic lineage and later, during terminal differentiation. Detection of H36-alpha 7 mRNA coincides with conversion of H36- myogenic precursor cells to H36+ cells. H36-alpha 7 mRNA is present in replicating myoblasts: expression increases upon terminal differentiation and is markedly reduced in developmentally defective myoblasts. In addition, H36-alpha 7 mRNA is not detected in C3H10T1/2 cells. It is in myotubes derived from myoblasts obtained by treatment of 10T1/2 cells with azacytidine or transfection with MRF4. Immunoblots and immunofluorescence demonstrate that the H36-alpha 7 chain is associated with integrin beta 1. Affinity chromatography demonstrates that H36-alpha 7 beta 1 selectively binds to laminin. The expression of H36-alpha 7 on secondary myoblasts during the development of the limb in vivo corresponds with the appearance of laminin in the limb, with the responsiveness of secondary myoblast proliferation to laminin, and with the onset of increased muscle mass, suggesting that H36-alpha 7 modulates this stage in limb development. We conclude that H36-alpha 7 is a novel alpha integrin laminin binding protein whose expression is developmentally regulated during skeletal myogenesis.

Amino Acid Sequence↗
PubMed1991

In vitro development of precursor cells in the myogenic lineage.

Expression of the muscle-specific integral membrane protein H36 and the intermediate filament protein desmin, detected by immunofluorescence, was used to identify cells at distinct stages in the skeletal myogenic lineage. These proteins were coordinately expressed in cultures of rat hindlimb myoblasts from 17- and 19-day fetuses and newborn pups, and in satellite cells from juveniles. Both H36+ and desmin+ cells were present in cultures from 13.5- and 15-day embryonic hindlimbs, but desmin expression was more prevalent: H36-/desmin+ myoblasts predominate during this early stage of development. H36 was not detected in Day 12 embryo hindlimb bud cells in vivo nor in cultures soon after plating. Initially, only 1% of the Day 12 limb bud cells expressed desmin. When these cells were serially passaged every 3-4 days, cells with all three possible myogenic phenotypes developed: that is, H36+/desmin-, H36+/desmin+, and H36-/desmin+ cells. There was a progressive increase in the frequency of H36+ cells, with 75% of cells positive by passage 6 (Day 27 in vitro). The maximum frequency of cells that expressed desmin occurred in passage 5 (Day 23 in vitro). These results demonstrate that precursors to the cells that express H36 and desmin are present in the 12-day embryo hindlimb bud and that the transition from H36-/desmin- precursors to cells with a myogenic phenotype can occur in vitro. MyoD1 and myogenin were not detected in these cells, suggesting that the initial expression of H36 and desmin in the myogenic lineage may precede and/or is independent of these regulatory proteins. The conversion of precursor cells in the 12-day limb bud to a more advanced stage of development serves to define additional cells in the myogenic lineage. The ability to monitor in vitro these stages of development affords the opportunity to study how they are regulated.

Animals↗
PubMed1990

Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly.

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.

Actins↗
PubMed1988

Replicating myoblasts express a muscle-specific phenotype.

During the terminal stage of skeletal myogenesis, myoblasts stop replicating, fuse to form multinucleate fibers, and express the genes that encode the proteins that convey contractile capacity. Because of this dramatic shift in proliferative state, morphology, and gene expression, it has been possible to readily identify and quantitate terminally differentiating myoblasts. In contrast, it is not clear whether the proliferating cells that give rise to postmitotic myoblasts are equally distinct in their phenotype and in fact whether distinct stages in skeletal myogenesis precede the onset of terminal differentiation. To address these questions, monoclonal antibodies and immunofluorescence microscopy were used to determine that replicating myoblasts from newborn rats do express a muscle-specific phenotype. To identify replicating cells, incorporation of 5-bromo-2'-deoxyuridine (BrdUrd) into DNA was assayed by using anti-BrdUrd antibody. The developmentally regulated, muscle-specific, integral membrane protein H36 and the intermediate-filament protein desmin were scored as markers of the myogenic phenotype. The percentage of BrdUrd+ (i.e., proliferative) cells among H36+ and desmin+ myoblasts was equal to the percentage of BrdUrd+ cells in the entire population, indicating that the expression of H36 and desmin is uniformly characteristic of replicating myoblasts. Inhibition of protein synthesis before and during growth in BrdUrd did not alter the frequency of desmin and H36 immunofluorescence in BrdUrd+ cells. Thus, desmin and H36 were present in the replicating myoblasts prior to the onset of growth in BrdUrd. These results were confirmed using H36+ cells selected by flow cytometry: these purified H36+ myoblasts replicate, express desmin, and differentiate. Similar results were obtained with mouse myoblasts. Desmin expression in these mammalian cells differs from that in chicken embryo myoblasts: only a small proportion of replicating chicken embryo myoblasts express desmin. That replicating mammalian myoblasts have a muscle-specific phenotype serves to define a distinct stage in myogenic development and a specific cell in the myogenic lineage. Further, it implies that there is a regulatory event activated during myogenesis that precedes terminal differentiation and that is required for expression of those genes whose products distinguish the replicating myoblast.

Animals↗
PubMed1987

A laminin substrate promotes myogenesis in rat skeletal muscle cultures: analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies.

Cells from newborn rat hindlimb show greatly enhanced myogenicity when grown on surfaces coated with poly-L-lysine followed by laminin (PLL/Lam) instead of the collagens routinely used. Coating with poly-L-lysine (PLL) alone or with PLL followed by collagen does not enhance myogenicity. Both myogenic and nonmyogenic cells, as distinguished by a monoclonal antibody specific for desmin, attach equally well to collagen- and laminin-coated surfaces, but there is a two- to five-fold increase in the number of myogenic cells on PLL/Lam by 72 hr, followed by increased myotube formation. To determine whether this increase in myogenic cells was a consequence of a selective increase in proliferation on PLL/Lam, incorporation of 5-bromodeoxyuridine into DNA followed by labeling with anti-BrdUrd antibody was used as an index of cell proliferation. The results indicate that desmin is expressed in replicating rat myoblasts, and that replication of myogenic cells is greatly enhanced on laminin compared to collagen. The rate of replication of nonmyogenic cells is the same on both substrates. Addition of 10 micrograms/ml laminin to the medium of cells seeded on PLL or collagen has no effect on myogenicity. We conclude that a laminin substrate enhances skeletal myogenesis in vitro by promoting selectively the replication of myoblasts. Cultures prepared from fetuses at 17 and 19 days gestation also show enhanced myogenicity when grown on PLL/Lam, while those from 15-day fetuses do not. Growth and development of fetal myoblasts on collagen were very poor, whereas myoblasts from the newborn rat do proliferate and differentiate on this substrate. Thus myogenic cells at different stages of fetal and neonatal development may require and respond to different extracellular environments. Myotube formation in the E63 clone of L8 rat myoblasts is inhibited by PLL/Lam.

Animals↗
PubMed1986

Endocytosis of alpha 2-macroglobulin is developmentally regulated during myogenesis.

A monoclonal antibody, H143, reacts with an intracellular antigen present and accumulated in E63 rat myoblasts. H143 is directed against a species-specific determinant on purified equine serum alpha 2-macroglobulin. Immunofluorescence analyses of differentiating myoblasts grown in horse serum demonstrate that the capacity to take up alpha 2-macroglobulin is stage-specific: the rapid uptake of alpha 2-macroglobulin characteristic of myoblasts ceases prior to their fusion to form multinucleate fibers (myotubes). Neither rat fibroblasts nor a developmentally defective mutant of E63 exhibit this change in alpha 2-macroglobulin uptake. The temperature and calcium requirements for the uptake of H143 antigen, and its accumulation as effected by lysosomotropic amines, indicate that alpha 2-macroglobulin is taken up by myoblasts via a developmentally regulated endocytic process. Electron microscopy using equine alpha 2-macroglobulin labeled with colloidal gold supports this finding.

Ammonium Chloride↗
PubMed1985

Remodeling of the myoblast membrane accompanies development.

We have prepared a library of cloned hybridomas that produce monoclonal antibodies reactive with the surface of E63 rat myoblasts. Using immunofluorescence analysis of antigens on single cells we have studied the expression of determinants at distinct stages of development. Conditions were established for quantitative photometry and were used to confirm the diversity in stage-specific expression that accompanies development. The remodeling of the myoblast membrane also involves stage-specific and transient changes in topography and aggregation of many antigens, and the period surrounding fusion is one of particular activity. The localization of antigens on the upper and attached surfaces of myogenic cells was often distinct, and quantitative and spatial stage-specific reorganizations of antigens differed with respect to these two surfaces. This polarity represents an additional level of complexity in the continuous remodeling of the muscle cell membrane. Comparisons of quantitative and topographic analyses of antigens on E63 cells with Rat-1 fibroblasts and developmentally defective (fu-) myoblasts indicate that the outer membranes of these nonmyogenic cells are distinct from differentiating myoblasts. One determinant, H36, is absent on Rat-1 cells and on all fu- lines tested, and undergoes interesting stage-specific changes in expression and topography.

Animals↗
PubMed1985

Expression of a developmentally regulated antigen on the surface of skeletal and cardiac muscle cells.

H36 is a species-specific, cell-surface antigen on differentiating newborn rat skeletal myoblasts and myogenic lines. This membrane antigen has been defined by a monoclonal antibody raised by the fusion of SP 2/0-Ag14 myeloma cells with spleen cells from mice immunized with myotubes derived from the myogenic E63 line. H36 antigen, isolated by immunoaffinity chromatography, is comprised of two polypeptides with apparent molecular weights of 98,000 and 117,000. Fluorescence photometry and radioimmunoassays have been used to follow quantitative and topographic changes in the H36 determinant during myogenesis. H36 is present at a basal level on replicating myoblasts; it increases on prefusion myoblasts and persists on myotubes. At or near the time of prefusion, it becomes concentrated between adjacent aligned myoblasts and localized on membrane "blebs". H36 is present on both skeletal and cardiac cells but absent from a variety of cells that include fibroblasts, neuronal cells, and smooth muscle. There are approximately 4 x 10(5) determinants per myoblast, and the Ka of the antibody is 3.8 x 10(8) liters/mol. The distributions of H36 on the top and attached surfaces of myoblasts and myotubes are distinct, which suggests localized specialization of these surfaces. H36 is an integral membrane component and upon cross-linking, it associates with the detergent-insoluble cytoskeletal framework. Inhibition of myogenesis by 5-bromodeoxyuridine or by calcium deprivation prevents the developmentally associated changes in the expression of H36. H36 is also absent or markedly reduced on the fu- and Ama102 developmentally defective mutant myoblast lines. We conclude that H36 is a muscle-specific, developmentally regulated cell-surface antigen that may have a role in myoblast differentiation and that can be used to determine the embryonic lineages of skeletal and cardiac muscle.

Animals↗
PubMed1971

Sodium transport by perfused giant axons of Loligo.

The sodium efflux from perfused squid giant axons has been studied using radioactive sodium, and the sufficient conditions for the maintenance of a potassium- and ouabain-sensitive sodium efflux have been established. The following were found.1. Axons extruded and then perfused with their own axoplasm had a sodium efflux which was sensitive to cyanide, potassium and ouabain and was thus similar to the efflux from intact axons.2. A method for replacing natural axoplasm into fibres previously perfused with artificial axoplasm was developed and used to establish an artificial perfusate that was not irreversibly toxic.3. Short perfusion (5 min) with a variety of artificial perfusates was then found to give fibres which had potassium- and ouabain-sensitive sodium effluxes when ATP was present in the perfusate.4. In the absence of ATP the sodium efflux was small and relatively insensitive to both external potassium and to ouabain.5. With ADP in the perfusate, fibres gave a sodium efflux which was ouabain-sensitive but was little affected by the removal of external potassium from the sodium-rich sea water bathing the fibres.6. The perfused fibres differed from intact fibres in having large ouabain-insensitive sodium effluxes.7. After very long perfusions (40-90 min), with the simple media containing ATP, the rate constant for sodium efflux from the fibres tended to be large and was relatively insensitive to potassium or to ouabain.8. Fibres refilled with natural axoplasm after long perfusion showed increased sensitivity to external potassium; refilled with dispersed axoplasm the sodium efflux tended to become very large.9. After very long perfusions with artificial axoplasms containing ATP, a potassium- and ouabain-sensitive sodium efflux was found to persist provided that dextran was present and the total osmotic pressure and the hydrostatic pressure of the perfusate were controlled. Under these conditions the sodium efflux resembled that from briefly perfused fibres. The necessary and sufficient conditions for the maintenance of sodium transport by perfused giant axons are discussed.

Adenosine Diphosphate↗
PubMed1969

The circular dichroism of suspensions of frog rod outer segments.

The circular dichroism (CD) of suspensions of frog rod outer segments has been measured. At wave-lengths between 600 and 400 nm1. The suspensions show a large positive CD increasing towards shorter wave-lengths and largely unaffected by bleaching. This probably arises, at least in part, from the preferential scattering of one form of circularly polarized light.2. There is a small reduction in CD on bleaching, slightly larger but similar in sign and wave-length dependence to that shown by pigment extracts. At wave-lengths between 250 and 220 nm3. The suspensions show a CD somewhat similar in sign, magnitude and wave-length dependence to that given by pigment extracts.4. On bleaching there is a reduction of the negative CD at 225 nm again similar to that which takes place when extracts are bleached. The observations suggest that configurational changes, of the kind detected by CD, occur in the bleaching of rhodopsin molecules whether they are present in solutions or in rods.

Animals↗