Ontogenetic limb bone scaling in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a potent mitogen which is required for normal development, particularly the development of the skeletal system, where the inhibition of FGF binding to its receptor results in various skeletal malformations. The present study employed a newly engineered line of FGF-2 transgenic mice to determine the effects of overexpressing FGF-2 on limb bone ontogeny. We collected radiographic and weight data longitudinally and obtained the length, proximal, distal, and minimum diaphyseal widths of the humerus, femur, and tibia. Because growth is nonlinear with respect to time, we used the Gompertz mathematical model to obtain parameters describing rate and timing for each individual for each measurement. Differences in the parameters due to genotype and sex were subsequently tested with ANOVA. Transgenic animals exhibited consistently shorter limb bones which were generally wider at the epiphyses than those of controls. Parameters of early growth, including initial size and proportional rate of growth, appeared to be most directly responsible for significant differences in final size; however, exponential decay of growth was also a marginally significant factor. There were no differences between the genotypes in body weight, indicating that the shape anomalies observed in transgenic mice were a direct result of the action of FGF-2 rather than a general runting phenomenon.

Wound healing in the transforming growth factor-beta-deficient mouse.

To investigate the role of transforming growth factor-beta(1) in tissue repair, we performed wound healing studies in the transforming growth factor-beta(1)-deficient mouse with targeted disruption of the transforming growth factor-beta(1) gene. Transforming growth factor-beta(1)-deficient mice exhibit no obvious developmental defects and are phenotypically normal until approximately 3 weeks of age when a severe wasting syndrome develops, accompanied by an overwhelming inflammatory response resulting in multisystem organ failure and death. Full-thickness 0.5 x 0.5 cm skin wounds were created on the backs of 10-day-old mice (wild type or heterozygous controls versus homozygous transforming growth factor-beta(1)-deficient mutants) and covered with a nonabsorbent dressing (OpSite). Serial wound measurements were made, and percentage of wound closure over time was determined. On day 10, wounds and liver were harvested for histologic and molecular analysis. Histologic scores were assigned (1 [no healing] to 12 [complete healing]) on the basis of granulation tissue formation, vascularity, collagen deposition, and epithelialization. Reverse transcription-polymerase chain reaction was performed to detect messenger RNA transcripts for transforming growth factor-beta(1), transforming growth factor-beta(2), platelet-derived growth factor A-chain and B-chain, interleukin-1beta and -6, and tumor necrosis factor-alpha in unwounded skin, day 10 wounds, and liver. No significant differences in wound closure were observed until day 10. Weight gain, however, was significantly decreased in the mutant animals as early as day 6. Histologic scores were significantly lower in the transforming growth factor-beta(1)-deficient mutants (5.4 +/- 0.6 versus 11.1 +/- 0.3, p < 0.01, Wilcoxon rank-sum test) and showed decreased granulation tissue formation, vascularity, collagen deposition, and epithelialization and a marked inflammatory infiltrate. As expected, transforming growth factor-beta(1) was expressed in controls but not mutants. Transforming growth factor-beta(2), platelet-derived growth factor A-chain and B-chain, and tumor necrosis factor-alpha were constitutively expressed in unwounded skin, day 10 wounds, and liver of both controls and mutants. Interleukin-1beta and -6, however, were induced after wounding. Early wound healing in the transforming growth factor-beta(1)-deficient mouse proceeds relatively normally because of upregulation or functional redundancy of other growth factors or possibly because of maternal rescue by means of transforming growth factor-beta(1) transmitted in milk. Loss of transforming growth factor-beta(1) regulation ultimately results in a marked inflammatory response, as evidenced by the histologic appearance of the wound and increased expression of the inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and 6). The severe wasting syndrome (marked by weight loss) undoubtedly has an adverse effect on wound healing.

Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives.

Transgenes in mice often exhibit different expression patterns in different transgenic lines. While the basis for this phenomenon is not understood, it is widely believed that the site at which the transgene becomes integrated into the mouse genome is a major factor in determining the pattern of expression. Most transgenic mice have been produced by microinjection of DNA into the male pronucleus, which results in integration of tandem arrays of the transgene at random chromosomal sites. In the experiments described in this report, electroporation of embryonic stem (ES) cells was used to place single copies of a lacZ transgene into either random sites or into the HPRT (hypoxanthine phosphoribosyl transferase) locus of the mouse genome. Expression of lacZ was assayed by histochemical staining for Escherichia coli beta-galactosidase activity in ES cells and in differentiated derivatives obtained by teratocarcinoma formation. Several of the randomly integrated cell lines expressed lacZ at high levels in a variety of cell types present in the tumours, but most notably in epithelial cells. Targeted cell lines with lacZ in opposite orientation to the direction of HPRT gene transcription also expressed well in epithelial cells, but the targeted cell lines did not express in a wider variety of cell types than some of the nontargeted cell lines. Targeted cell lines transcribing lacZ in the same orientation as HPRT transcription did not express high levels of lacZ in any differentiated cell type. Analysis of transcripts suggested that this orientation effect may have been the result of transcriptional interference perpetrated by the HPRT gene promoter.

The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium.

The in vitro developmental potential of mouse blastocyst-derived embryonic stem cell lines has been investigated. From 3 to 8 days of suspension culture the cells form complex embryoid bodies with endoderm, basal lamina, mesoderm and ectoderm. Many are morphologically similar to embryos of the 6- to 8-day egg-cylinder stage. From 8 to 10 days of culture about half of the embryoid bodies expand into large cystic structures containing alphafoetoprotein and transferrin, thus being analagous to the visceral yolk sac of the postimplantation embryo. Approximately one third of the cystic embryoid bodies develop myocardium and when cultured in the presence of human cord serum, 30% develop blood islands, thereby exhibiting a high level of organized development at a very high frequency. Furthermore, most embryonic stem cell lines observed exhibit similar characteristics. The in vitro developmental potential of embryonic stem cell lines and the consistency with which the cells express this potential are presented as aspects which open up new approaches to the investigation of embryogenesis.

Comparison of M-line and other myofibril components during reversible phorbol ester treatment.

The events occurring during phorbol ester mediated destruction of myofibrils in differentiated muscle cells were followed at the fluorescence and electron microscope levels using antibodies which bind troponin-T, a newly discovered 185 000 dalton M-line protein called myomesin and muscle type creatine kinase. The following series of events is proposed. Within one day of phorbol ester treatment, Z-bands and thin filaments, including troponin-T, are absent from many myofibrils resulting in the rapid loss of longitudinal and lateral alignment. A-bands become randomly oriented and clustered into ever smaller compartments within the rounding, myosac-like, multinucleated cells until after 3 days of treatment they too disappear. The M-line proteins are always present in existing A-bands. These results suggest that the Z-band and associated structures are responsible for the maintenance of alignment and the lateral register of myofibrils, whereas the M-line is responsible for the structural integrity of the A-band. When phorbol ester is removed, the cells revert to a myotube morphology and within 2 to 3 days are filled with myofibrils. A comparison of the appearance of troponin-T and the 185 000 dalton myomesin in the recovery period to their appearance during normal myofibrillogenesis reveals that these proteins are more temporally co-ordinated during myofibrillogenesis than in the phorbol ester experimental system.

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies.

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.

Novel staining pattern of skeletal muscle M-lines upon incubation with antibodies against MM-creatine kinase.

Incubation of chicken skeletal muscle fibers with an excess of anti-M-creatine kinase (CK) immunoglobulin G and an excess of anti-M-CK Fab fragments leads to heavy decoration of the M-line (Wallimann, T., D.C. Turner, and H.M. Eppenberger, 1977, J. Cell Biol. 75:297-317) and to removal of the electron-dense M-line structure (Walliman, T., G. W. Pelloni, D.C. Turner, and H.M. Eppenberger, 1978, Proc. Natl. Acad. Sci. USA., 75:4296-4300), respectively. On the other hand, incubation with low concentrations of monovalent anti-M-CK Fab did not extract but rather decorated the M-line, giving rise to a distinct two-line staining pattern. A similar double-line staining pattern, although less pronounced, was also observed within the M-line of paraformaldehyde-prefixed myogenic cells, which after permeabilization were incubated with low concentrations of divalent anti-M-CK antibody. In both cases, the two decorated lines appearing in the middle of the A-band were spaced axially 42-44 nm apart and correspond most likely to the two M4 and M4' m-bridge rows described by Sjöström and Squire (1977, J. Mol. Biol., 109:49-68; 1977, J. Microscopy., 111:239-278). It is concluded that the muscle-specific form of creatine kinase, MM-CK, contributes mainly to the electron density of these M4 and M4' m-bridges within the M-line structure. This specific labeling pattern is a further demonstration that CK is an integral part of the M-line.

The contribution of cell death to medium-released fractions of cell cultures.

Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [3H]thymidine and by subsequently measuring the release of label into complete culture medium or serum- and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [3H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the 3H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account.

The effects of Con A on cell surface shedding in cell cultures.

A cell surface immobilizing concentration of Con A inhibits the shedding of [3H]fucose-containing glycoproteins from the surface of chick embryonic leg and breast muscle cell cultures and cultures of the rat skeletal muscle cell line L6. The Con A-induced inhibition of shedding is much less in the mouse fibroblast cell line 3T3. In all 4 cell types the lectin inhibits the shedding of some fucosyl-glycoproteins more than others, especially those of a lipid-containing fraction which is excluded in Biogel A-5m chromatography. This differential nature of the Con A effect is not changed by the cytoskeletal disruptors cytochalasin B or colchicine. Con A causes an increase in the amount of trypsin-sensitive surface fucosylglycoprotein in the cell surface and appears to decrease the overall amount of cell surface degradation suggesting the inhibition of shedding caused by Con A is not due to an increase in internalization and degradation. The data suggest that some shedding may occur at specific cell surface sites to which surface materials must laterally migrate.

Myoblast stored myosin heavy chain transcripts are precursors to the myotube polysomal myosin heavy chain mRNAs.

Radioactively labeled myosin heavy chain messenger ribonucleic acid (MHC mRNA) synthesized during the pre-fusion stage of chick embryo breast muscle cell culture is transferred from messenger ribonucleic acid proteins (mRNPs) to the polysomal MHC mRNA during the period of rapid increase in the rate of MHC synthesis (mid-to late-fusion). This transfer constitutes a major contribution to the rate of incorporation of 3H-labeled transcripts into polysomal MHC mRNA at this time. As the increase in the rate of MHC synthesis levels off (late-to post-fusion) the contribution to the rate of incorporation of 3H-labeled transcripts into polysomal MHC mRNA from newly synthesized transcripts increases until it becomes predominant. In vivo, the level of MHC mRNP increases during early stages of embryonic development and then decreases when MHC synthesis and the level of polysomal MHC mRNA has been shown to increase.

Cell surface turnover and shedding of fucosyl-glycoprotein in L6 cells.

The disappearance of electrophoretically identifiable 3H-fucosylated glycoproteins from the cell surface of L6 cells is random and follows monophasic kinetics with a half-life of 9 hours. Approximately 47% and 53% of that loss is due to shedding and to degradation, respectively. Shedding also occurs with monophasic kinetics. Most of the electrophoretically identifiable-shed fucosyl-glycoproteins are released with homogeneous rates but do not represent a random sampling of the surface.

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.