SEARCH

SEARCH BY CITATION

Keywords:

  • extracellular matrix;
  • fibroblast growth factor 2;
  • glypican;
  • muscle;
  • turkey

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The heparan sulfate proteoglycan, glypican-1, is a low affinity receptor for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and an inhibitor of differentiation. Heparan sulfate proteoglycans like glypican-1 are required for FGF2 to transduce an intracellular signal. Understanding the role of glypican-1 in the regulation of FGF2-mediated signaling is important in furthering the understanding of the biological processes involved in muscle development and growth. In the current study, a turkey glypican-1 expression vector construct was transfected into turkey myogenic satellite cells resulting in the overexpression of glypican-1. The proliferation, differentiation, and responsiveness to FGF2 were measured in control and transfected cell cultures. The overexpression of glypican-1 in turkey myogenic satellite cells increased both satellite cell proliferation and FGF2 responsiveness, but decreased the rate of differentiation. The current data support glypican-1 modulation of both proliferation and differentiation through an FGF2-mediated pathway.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Skeletal muscle myogenesis is a complex process that involves muscle cell proliferation, migration, adhesion, fusion to form multinucleated myotubes, and further differentiation into muscle fibers (Swartz et al. 1994). This process is precisely regulated, in part, through the muscle cell interacting with the extrinsic or extracellular environment surrounding the cells. The extracellular environment or matrix (ECM) is composed of protein and polysaccharides synthesized by the cells (Scott 1995). The interaction of myoblasts with the ECM is required for cell fusion leading to the formation of multinucleated myotubes (Melo et al. 1996).

Proteoglycans are one component of the ECM and represent a diverse family of glycosylated proteins located either extracellularly or associated with the cell surface that contain a core protein with covalently attached glycosaminoglycans (Hardingham & Fosang 1992). Glycosaminoglycan chains attached to the core protein include chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate.

The cell surface heparan sulfate proteoglycan families, the syndecans and glypicans, function as low-affinity receptors for fibroblast growth factor 2 (FGF2). FGF2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation (Dollenmeier et al. 1981). Muscle cell responsiveness to FGF2 will therefore have a significant impact on muscle growth. Heparan sulfate is required for FGF2 to transduce an intracellular signal (Rapraeger et al. 1991; Yayon et al. 1991). The glycosylphosphatidylinositol cell surface anchored heparan sulfate proteoglycan, glypican-1, has been shown to increase in expression during muscle differentiation (Brandan et al. 1996; Liu et al. 2004). There are six identified forms of glypican, glypican-1 through 6, of which only glypican-1 has been identified in skeletal muscle. The increase in glypican-1 expression when multinucleated myotubes are forming has been hypothesized to function by sequestering FGF2. This would permit the process of differentiation to proceed without the inhibitory effects of FGF2. The role of glypican-1 to sequester FGF2 is only based on its expression, not biological data. It was the goal of the present study to begin to address the biological function of glypican-1 in the regulation of muscle cell proliferation and differentiation, by transfecting muscle cell cultures with a glypican-1 expression vector construct and measuring the effect on proliferation, differentiation, and responsiveness to FGF2.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Turkey myogenic satellite cells

Satellite cells were isolated from the pectoralis major muscle of 7-week-old male F-line turkeys as described in Velleman et al. (2000). The F-line was selected long-term for increased 16 week body weight.

Total RNA extraction and cDNA synthesis

Total RNA was extracted from the cultured satellite cells with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's procedure. The cDNA from the total RNA was synthesized using Moloney murine leukemia virus reverse transcriptase (M-MLV; Promega, Madison, WI, USA) based on the protocol described by the manufacturer. The reverse transcription (RT) reaction was performed in a 25 µL volume. The RNA-primer mix (1 µL oligo d(T)20 (50 µm) (Ambion, Austin, TX, USA), 1 µg of total RNA, nuclease-free water up to 13.5 µL) was heated at 70°C for 5 min. This mixture was then incubated immediately on ice for 2 min and 11.5 µL of reaction mix (5 µL of 5× first-strand buffer (250 mm Tris-HCl pH 8.3, 375 mm KCl, 15 mm MgCl2, 50 mm dithiothreitol; Invitrogen), 5 µL 10 mm deoxynucleoside triphosphate mix (Promega), 0.25 µL RNasin (40 U/µL; Promega), 1 µL M-MLV (200 U/µL) and nuclease free H2O up to 11.5 µL) was added. The reaction mixture was incubated at 55°C for 60 min, and then heated at 70°C for 15 min to stop the reaction. The synthesized cDNA was diluted with 25 µL nuclease-free water before performing the real-time quantitative polymerase chain reaction (PCR).

Construction of the turkey glypican-1 expression vector

The cDNA containing the entire turkey glypican-1 coding sequence (1650 bp) was amplified from total RNA isolated from 18 day embryonic turkey F-line pectoral muscle using RT–PCR. The RT reaction was conducted as described above. The PCR primers were designed from the published turkey glypican sequence (GenBank accession number AY551002). The forward PCR primer was 5′-CCG CTC GAG ATG CGT TTC TTC CCG TGG GGA TTT-3′ which added a XhoI restriction site (indicated in bold) to the 5′ end, and the backward PCR primer was 5′-TGC TCT AGA TTA CCG CCA CAA GTG CTG CAC-3′ which added a XbaI restriction site (indicated in bold) to the 3′ end. The PCR reaction was performed in a 50 µL total volume reaction mixture containing 2 µL of the RT reaction, 0.2 µm of each forward and backward primer, 5 µL AccuPrime Pfx Reaction Mix (Invitrogen), and 1 U Accuprime Pfx DNA Polymerase (Invitrogen). Amplification consisted of an initial denaturation at 95°C for 2 min, and 35 cycles of 15 s at 95°C, 30 s at 55°C, and 2 min at 68°C followed by 10 min at 68°C. The amplified product was gel-purified with QIAquick Gel Extraction kit (Qiagen, Valencia, CA, USA) and digested with 20 U of XhoI (Invitrogen) and XbaI (Qiagen) overnight at 37°C. Likewise, 2 µg pCMS-EGFP vector (BD Biosciences Clontech, Palo Alto, CA, USA) was digested with XhoI and XbaI for 4 h at 37°C. Both restriction-digested glypican-1 and pCMS-EGFP vector were gel-purified with QIAquick Gel Extraction kit. The ligation reaction was set up in a 20 µL total volume which included 2 µL 10× T4 ligation buffer (300 mm Tris-HCl, pH 7.8, 100 mm MgCl2, 100 mm dithiothreitol and 10 mm adenosine triphosphate) (Promega), 3 U of T4 DNA ligase (Promega), 50 ng restriction-digested pCMS-EGFP vector and 100 ng restriction-digested glypican-1. The ligation mixture was incubated at 4°C overnight. Six microliters of the ligation reaction was used to transform 45 µL subcloning efficiency DH5α chemically competent Escherichia coli (Invitrogen) according to the manufacturer's instruction. Luria-Bertani (LB)-ampicillin agar plate (LB broth pH 7.0: 1% bacto-tryptone, 0.5% bacto-yeast extract, and 1% NaCl + 100 µg/mL ampicillin + 1.5% bacto-agar (all reagents from Fisher Scientific Pittsburgh, PA, USA)) was used to select the clones. The positive clones were grown overnight at 37°C in 5 mL of LB broth containing 100 µg/mL ampicillin to expand the plasmids. The plasmids were isolated using a QIAquick Miniprep kit (Qiagen) and the glypican-1 insert of the plasmid was confirmed by DNA sequencing.

Real-time quantitative polymerase chain reaction

The real-time quantitative PCR was performed using a DyNAmo Hot Start SYBR Green qPCR kit (MJ Research, Las Vegas, NV, USA) as described in Liu et al. (2005). Primers (Qiagen) used for the amplification of glypican-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed from published turkey sequences as listed in Table 1. Following the reaction assembly, plates were put into the DNA Engine Opticon 2 real-time system (MJ Research). The cycling program consisted of denaturation (95°C for 15 min), followed by amplification and quantitation (34 cycles of 94°C for 20 s, 54°C for 20 s and 72°C for 20 s, with a single fluorescence measurement at the end of 72°C of each cycle) and a final extension of 72°C for 5 min. The melting curve program was 52°C to 95°C, 0.2°C/read, and a 1 s hold. The final PCR products were sequenced and analyzed on a 1.5% agarose gel to check for amplification specificity. Standard curves were constructed for glypican-1 and GAPDH with serial dilutions of purified PCR products from each gene. The PCR products were purified by agarose gel electrophoresis using a QIAquick gel extraction kit (Qiagen). All the sample concentrations fell within the values of the standard curves. The amount of sample cDNA for each gene was interpolated from the corresponding standard curve. The expression of glypican-1 was normalized to GAPDH expression.

Table 1.  Primer sequences for real-time polymerase chain reaction
PrimerSequenceProduct size
  • Primer sequences were designed from the following GenBank accession numbers: turkey glypican-1 (GPC-1), AY551002; turkey glyceraldehyde-3-phosphate dehydrogenase (GAPDH), U94327.

GPC-15′-CTTGTCGCTGTGGCAGATCGG-3′ (Forward)176 bp
5′-CTGCTGGAGCCTTTTGTGCTGA-3′ (Backward) 
GAPDH5′-GAGGGTAGTGAAGGCTGCTG-3′ (Forward)200 bp
5′-CCACAACACGGTTGCTGTAT-3′ (Backward) 

Proliferation assay

Satellite cells were plated at a density of 37 500 cells per well in gelatin-coated 16 mm well plates (Sarstedt, Newton, NC, USA). The cultures were plated in Dulbecco's Modified Eagle's Medium (DMEM; Atlanta Biologicals, Lawrenceville, GA, USA) containing 10% chicken serum (Invitrogen), 5% horse serum (Invitrogen) and 1% antibiotic/antimycotic (Invitrogen) and grown in a 37°C 95% air/5% CO2 incubator (Fisher Scientific). Twenty-four hours after plating the cultures were transfected with the glypican-1 expression vector and just the pCMS-EGFP vector. The plasmid DNA used in the transfections was purified with a Bio 101 Systems RPM Spin Midi kit (Q-BIOgene, Irvine, CA, USA). The cultures were transfected using Clonfectin (BD Biosciences Clontech) according to the manufacturer's recommended conditions with 2 µg of plasmid DNA and 4 µg Clonfectin. The cell cultures were incubated with the transfection solution for 2 h at 37°C in a 95% air/5% CO2 incubator. After the incubation, the transfection solution was removed and the cells were washed with 37°C DMEM. Growth medium (McCoy's 5A (Atlanta Biologicals), 15% chicken serum, 10% horse serum, and 0.1% antibiotic/antimycotic) was then added to the cultures and grown at 37°C in a 95% air/5% CO2 incubator. Every 24 h after the transfection for 96 h, plates were removed, wells were rinsed with phosphate-buffered saline (PBS), and stored at −70°C until assayed. Proliferation was measured by the DNA content of the wells using cultures that had a 60–65% transfection efficiency. The DNA was analyzed using Hoechst 33258 fluorochrome (Sigma, St Louis, MO, USA) by the method of McFarland et al. (1995) adapted from the fluorometric procedure described by Rago et al. (1990) using double-stranded calf thymus DNA as the standard. The DNA concentration was measured on a Fluoroskan Ascent FL (ThermoElectron Co, Waltham, MA, USA).

Responsiveness to fibroblast growth factor 2

The F-line turkey satellite cells were plated at a density of 15 000 cells in gelatin-coated 16 mm plates and grown for 24 h in DMEM containing 10% chicken serum, 5% horse serum, without antibiotics or antimycotics in a 37°C 95% air/5% CO2 environment. After 24 h the cultures were transfected with either the glypican-1 expression vector or pCMS-EGFP using Invitrogen's Optifect Reagent with 1 µg of plasmid DNA per culture well according to the manufacturer's protocol. Plasmid DNA used in the transfections was purified with a Bio 101 Systems RPM Spin Midi kit. After the transfection incubation serum-free defined media (McFarland et al. 1991) was added to the cell cultures containing 0, 0.5, 1.0, 5.0, and 10.0 ng/mL of FGF2 (Pepro Tech, Rocky Hill, NJ, USA). The media was changed daily for 72 h. At 96 h post-transfection, the plates were rinsed with PBS, air dried, and stored at −70°C until analysis. FGF2 responsiveness was measured by the DNA content of the wells in cultures with a 60–65% transfection efficiency. The DNA was analyzed using Hoechst 33258 fluorochrome by the method of McFarland et al. (1995) adapted from the fluorometric procedure described by Rago et al. (1990) using double-stranded calf thymus DNA as the standard. The DNA concentration was measured on a Fluoroskan Ascent FL.

Differentiation assay

The F-line turkey satellite cells were plated at a density of 15 000 cells in gelatin-coated 16 mm plates and grown for 24 h in DMEM containing 10% chicken serum, 5% horse serum, without antibiotics or antimycotics in a 37°C 95% air/5% CO2 environment. The transfection was performed by the same method as for the FGF responsiveness assay. After the transfection incubation, the cell cultures were incubated at 37°C in a 95% air/5% CO2 incubator in McCoy's 5A medium containing 10% chicken serum, and 5% horse serum. The medium was changed daily until differentiation was induced at 72 h post-transfection when the cells reached 65% confluency by changing the medium to DMEM containing 3% horse serum, 0.01 mg/mL porcine gelatin (Invitrogen) and 1.0 mg/mL bovine serum albumin (Sigma). At each sampling time, the plates were removed from the incubator, washed with PBS, dried, and stored at −70°C until analysis. Differentiation was determined by measuring the muscle specific creatine kinase protein levels by the procedure of Florini (1989) in samples with a 60–65% transfection efficiency.

Statistical analysis

To estimate differences in the expression of glypican-1 to that of the control transfected cells differences between means were evaluated using a Student's t-test. Differences were considered significant if P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The overexpression of glypican-1 in the transfected F-line turkey myogenic satellite cells was confirmed by real-time PCR amplification. The transfected cells were analyzed for glypican-1 expression 48 h after the transfections. The glypican-1 transfected cells had a significant increase in glypican-1 expression compared to the controls (Fig. 1).

image

Figure 1. Real-time polymerase chain reaction analysis of glypican-1 RNA expression in F-line male turkey satellite cell cultures. The RNA expression was measured 48 h after the transfection of the satellite cells with the glypican-1 construct (GPC-1) or the control vector without an insert (Control). The error bar represents the standard error. *Indicates a significant difference (P < 0.05).

Download figure to PowerPoint

The glypican-1 transfected and control cells were cultured and assayed for DNA accretion as an indication of proliferation. Comparison of the glypican-1 transfected cell cultures to the control showed at 48 and 96 h post-transfection that proliferation was increased in the glypican-1 transfected cells (Fig. 2). Corresponding to the increase in proliferation was an overall trend of elevated responsiveness of the glypican-1 transfected cells to FGF2 (Fig. 3).

image

Figure 2. Proliferation of F-line turkey satellite cells transfected with the glypican expression vector construct (GPC-1) or the control vector without an insert (Control). The error bar represents the standard error. *Indicates a significant difference (P < 0.05).

Download figure to PowerPoint

image

Figure 3. Responsiveness of F-line turkey satellite cells transfected with glypican-1 expression vector construct (GPC-1) or the control vector with an insert (Control) to increasing concentrations of fibroblast growth factor 2 (FGF2). The error bar represents the standard error. *Indicates a significant difference (P < 0.05).

Download figure to PowerPoint

After differentiation was induced in the control and glypican-1 cell cultures, differentiation was assayed by creatine kinase levels. Beginning at 48 h of differentiation, the cell cultures overexpressing glypican-1 began to show a decrease in creatine kinase levels indicating a reduction in differentiation (Fig. 4) The reduction in differentiation rate at 96 h in both the control and glypican transfected cells is likely due to the detachment of the cells from the culture well.

image

Figure 4. Differentiation of F-line turkey satellite cells transfected with glypican-1 expression vector construct (GPC-1) or the control vector without an insert (Control). The error bar represents the standard error of the mean. *Indicates a significant difference (P < 0.05).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Satellite cells are myogenic cells located between the basement membrane and plasma membrane of muscle fibers (Mauro 1961), and are the major contributors to postnatal muscle growth and the regeneration of muscle (Moss & LeBlond 1971). Heparan sulfate proteoglycans including the syndecans and glypicans are low-affinity receptors for FGF2. FGF2 stimulates skeletal muscle cell proliferation but inhibits differentiation (Dollenmeier et al. 1981). The heparan sulfate proteoglycans are differentially expressed during both in vivo and in vitro muscle development (Liu et al. 2004; Liu et al. 2006). Functions for syndecan-1 through 4 and glypican-1 have been postulated based on their expression pattern (Brandan et al. 1996; Larra’n et al. 1997; Cornelison et al. 2001; Casar et al. 2004; Liu et al. 2004; Liu et al. 2006), but direct evidence for their precise biological function has not been reported.

To begin to address the biological function of glypican-1 in the regulation of muscle cell proliferation and differentiation, a glypican-1 expression vector construct was made and transfected into muscle cell cultures to measure the effect on proliferation, differentiation and responsiveness to FGF2. The results from the present study showed that the overexpression of glypican-1 in turkey myogenic satellite cell cultures enhances proliferation. The increase in proliferation or growth of the satellite cells is likely due to the elevation in FGF2 responsiveness. During differentiation, the transfected cells overexpressing glypican-1 exhibited a reduction in their differentiation rate compared to the controls.

In vitro studies have shown increases in the expression of glypican-1 during differentiation (Brandan et al. 1996; Liu et al. 2006). These expression data suggest that glypican-1 may play a role during muscle differentiation. However, the regulation of muscle growth may not only be affected by the expression level of the glypican-1 but also the biological form. Glypican-1 can be associated with the cell surface as an insoluble membrane-associated heparan sulfate proteoglycan or be shed from the cell surface and exist as a soluble form. Brandan et al. (1996) reported in C2C12 mouse muscle cells, glypican-1 was released from the cell surface into the incubation medium. Based on this observation, Brandan et al. (1996) have postulated that the enhanced expression and shedding of glypican during muscle cell differentiation may be part of a cellular mechanism to sequester FGF2 to permit differentiation. Heparan sulfate is required for the stable binding of FGF2 to its tyrosine kinase receptor forming a FGF-receptor signaling complex (Rapraeger et al. 1991). Glypican in the membrane-associated form is likely to be able to present FGF2 to its receptor and in the soluble form FGF2 will still be able to bind to the glypican heparan sulfate proteoglycan chains, but not interact with its receptor. Hence, the soluble form of glypican would suppress FGF2 signaling and differentiation would proceed.

The results from the current study support a role for glypican-1 in the proliferation of muscle cells. The transfection of the glypican-1 likely increased the amount of cell surface or the insoluble form of glypican which would increase FGF signaling if glypican was functioning as a co-receptor for FGF2. In a previous study (Velleman et al. 2004), transfection of glypican-1 into F-line satellite cells resulted in the formation of larger myotubes than control muscle cell cultures which may be the result of an increase in cell number during proliferation. If more muscle cells are present due to an increase in proliferation then upon stimulating differentiation, more or larger myotubes would form. In fast growing mice, prolonged myoblast proliferation has been thought to contribute to muscle fiber hyperplasia at birth (Summers & Medrano 1997).

Based on expression profile information, and not direct biological data, it has been speculated that glypican may sequester FGF2 to permit differentiation to proceed as glypican expression increases with muscle differentiation in vitro (Brandan & Larraín 1998). The results from the present study showed a decrease in differentiation rate in the transfected muscle cell cultures. This may result from more of the glypican-1 in the transfected cells remaining associated with the cell surface and not being shed. Brandan et al. (1996), using C2C12 mouse muscle cell cultures, observed that as differentiation proceeded, glypican was released from the cell surface. Future studies will need to examine the ratio of insoluble to soluble glypican and the interaction with FGF2 to ascertain more information with regard to the role of glypican in muscle development and growth.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Salary and research support to S. G. V. was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, research support from the Cooperative State Research, Education, and Extension Service, US Department of Agriculture under Agreement number 2003-35206-13696, and to S. G. V. and D. C. M. research support from the Midwest Poultry Research Consortium.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Brandan, E., Carey, D. J., Larraín, J., Melo, F. & Compso, A. 1996. Synthesis and processing of glypican during differentiation of skeletal muscle cells. Eur. J. Cell Biol. 71, 170176.
  • Brandan, E. & Larraín, J. 1998. Heparan sulfate proteoglycans during terminal skeletal muscle differentiation: possible functions and regulation of their expression. Basic Appl. Myol. 8, 107113.
  • Casar, J. C., Cabello-Verrugio, C., Olguin, H., Aldunate, R., Inestrosa, N. C. & Brandan, E. 2004. Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation. J. Cell Sci. 117, 7384.
  • Cornelison, D. D., Filla, M. S. & Stanley, H. M. 2001. Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev. Biol. 239, 7994.
  • Dollenmeier, P., Turner, D. C. & Eppenberger, H. M. 1981. Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium. Exp. Cell Res. 135, 4761.
  • Florini, J. R. 1989. Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nm. Anal. Biochem. 182, 399404.
  • Hardingham, T. E. & Fosang, A. J. 1992. Proteoglycans: many forms and many functions. FASEB J. 6, 861870.
  • Larraín, J., Cizmeci-Smith, G., Troncoso, V., Stahl, R. C., Carey, D. J. & Brandan, E. 1997. Syndecan-1 expression is down-regulated during myoblast terminal differentiation. Modulation by growth factors and retinoic acid. J. Biol. Chem. 272, 18 41818 424.
  • Liu, X., McFarland, D. C., Nestor, K. E. & Velleman, S. G. 2004. Developmental regulated expression of syndecan-1 and glypican in pectoralis major muscle in turkeys with different growth rates. Dev. Growth Diff. 46, 3751.
  • Liu, C., McFarland, D. C., Nestor, K. E. & Velleman, S. G. 2006. Differential expression of membrane-associated heparan sulfate proteoglycans in the skeletal muscle of turkeys with different growth rates. Poult. Sci. in press.
  • Liu, C., McFarland, D. C. & Velleman, S. G. 2005. Effect of genetic selection on MyoD and myogenin expression in turkeys with different growth rates. Dev. Growth. Diff. 84, 376384.
  • Mauro, A. 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, 493495.
  • McFarland, D. C., Pesall, J. E., Gilkerson, K. K., Ye, W. V., Walker, J. S. & Wellenreiter, R. 1995. Comparison of in vitro properties of satellite cells derived from the pectoralis major and biceps femoris of growing turkeys. Basic Appl. Myol. 5, 2731.
  • McFarland, D. C., Pesall, J. E., Norberg, J. M. & Dvoracek, M. A. 1991. Proliferation of the turkey myogenic satellite cell in a serum-free medium. Comp. Biochem. Physiol. 99A, 163167.
  • Melo, F., Carey, D. J. & Brandan, E. 1996. Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression. J. Cell. Biochem. 62, 227239.
  • Moss, F. P. & LeBlond, C. P. 1971. Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170, 421435.
  • Rago, G., Mitchen, J. & Wilding, G. 1990. DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal. Biochem. 191, 3134.
  • Rapraeger, A., Krufka, A. & Olwin, B. B. 1991. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science 252, 17051708.
  • Scott, J. E. 1995. Extracellular matrix, supramolecular organisation and shape. J. Anat. 187, 259269.
  • Summers, P. J. & Medrano, J. F. 1997. Delayed myogenesis associated with muscle fiber hyperplasia in high-growth mice. Proc. Soc. Exp. Biol. Med. 214, 380385.
  • Swartz, D. R., Lim, S.-S., Fassel, T. & Greaser, M. L. 1994. Mechanisms of myofibril assemble. Recip. Meat Conf. Prof. 47, 141153.
  • Velleman, S. G., Liu, X., Coy, C. S. & McFarland, D. C. 2004. Effects of syndecan-1 and glypican on muscle cell proliferation and differentiation: Implications for possible functions during myogenesis. Poult. Sci. 83, 10201027.
  • Velleman, S. G., Liu, X., Nestor, K. E. & McFarland, D. C. 2000. Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates. Comp. Biochem. Physiol. 125A, 503509.
  • Yayon, A., Klagsburn, M., Esko, J. D., Leder, P. & Oritz, D. M. 1991. Cell surface heparin-like molecules are required for binding of basic fibroblast growth factors to its high affinity receptors. Cell 64, 841848.