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Poult Sci 2009. 88:265-275. doi:10.3382/ps.2008-00234
© 2009 Poultry Science Association
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GENETICS

Effect of transforming growth factor-β1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken1

X. Li and S. G. Velleman2

Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster 44691

2 Corresponding author: velleman.1{at}osu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During skeletal muscle development, transforming growth factor-β1 (TGF-β1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-β1 signal is carried by Smad proteins into the cell nucleus, inhibiting the expression of key myogenic regulatory factors including MyoD and myogenin. However, the molecular mechanism by which TGF-β1 inhibits muscle cell proliferation and differentiation has not been well documented in vivo. The present study investigated the effect of TGF-β1 on in vivo skeletal muscle growth and development. A chicken line, Low Score Normal (LSN) with reduced muscling and upregulated TGF-β1 expression, was used and compared to a normal chicken line. The injection of TGF-β1 at embryonic day (ED) 3 significantly reduced the pectoralis major (p. major) muscle weight in the normal birds at 1 wk posthatch, whereas no significant difference was observed in the LSN birds. The difference between normal and LSN birds in response to TGF-β1 is likely due to different levels of endogenous TGF-β1 where the LSN birds have increased TGF-β1 expression in their p. major muscle at both 17 ED and 6 wk posthatch. Smad3 expression was reduced by TGF-β1 from 10 ED to 1 wk posthatch in normal p. major muscle. Unlike Smad3, Smad7 expression was not significantly affected by TGF-β1 until posthatch in both normal and LSN p. major muscle. Expression of MyoD was reduced 35% by TGF-β1 during embryonic development in normal p. major muscle, whereas LSN p. major muscle showed a delayed decrease at 1 d posthatch in MyoD expression in response to the TGF-β1 treatment. Myogenin expression was reduced 29% by TGF-β1 after hatch in normal p. major muscle. In LSN p. major muscle, TGF-β1 treatment significantly decreased myogenin expression by 43% at 1 d posthatch and 32% at 1 wk posthatch. These data suggested that TGF-β1 reduced p. major muscle growth by inhibiting MyoD and myogenin expression during both embryonic and posthatch development. Furthermore, TGF-β1 also reduced the expression of the cell adhesion receptor β1 integrin subunit during embryonic and posthatch muscle growth in normal and LSN chickens. Therefore, the reduction of β1 integrin in response to TGF-β1 is also associated with decreased posthatch muscle growth. The results from this study indicate that TGF-β1 inhibits skeletal muscle growth by regulating MyoD and myogenin expression. These data also suggest that a β1 integrin-mediated alternative pathway is likely involved in the TGF-β1-induced reduction of muscle growth.

Key Words: myogenin • MyoD • muscle • Smads • transforming growth factor-β1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Skeletal muscle development is a highly organized process involving the precise regulation of numerous developmental events. These biological processes include, but are not limited to, cell adhesion, cell migration, and cell-to-cell interactions. The presumptive myoblasts are regulated by positive and negative signals from the extracellular environment. The presumptive myoblasts migrate from the mesodermal somites to the appropriate region and become committed myoblasts. The committed myoblasts proliferate, align with each other, and fuse to form multinucleated myotubes (Chargé and Rudnicki, 2004).

Skeletal muscle growth occurs in 2 phases: hyperplasia and hypertrophy. Hyperplasia is defined as the increase in muscle cell number, whereas hypertrophy refers to the increase in muscle fiber size. The number of embryonic myofibers is set around birth or hatch (Smith, 1963). Postnatal muscle growth is regulated by quiescent, undifferentiated cells called satellite cells. During the period of postnatal muscle growth, satellite cells proliferate, differentiate, and fuse with adjacent muscle fibers or with other satellite cells. The additional nuclei are then added to the muscle fibers, ultimately leading to increased muscle mass through increased protein synthesis resulting in muscle fiber hypertrophy (Allen et al., 1979; Allen and Goll, 2003). Both hyperplasia and hypertrophy are regulated by interactions between muscle cells and their extracellular matrix (ECM; Velleman, 1999).

Growth factors secreted outside the cell into the ECM play a pivotal role in regulating cell proliferation and differentiation. Transforming growth factor-β1 (TGF-β1), a multifunctional regulator of cell growth and differentiation (Roberts and Sporn, 1985), has been shown to inhibit both myoblast proliferation and differentiation (Allen and Boxhorn, 1987). During myoblast proliferation and differentiation, the expression of the myogenic regulatory factors are increased and induce a set of muscle-specific genes, resulting in myoblast proliferation and fusion to form multinucleated myotubes (Florini et al., 1991). Myogenic regulatory factors, including MyoD, Myf5, myogenin, and MRF4, are basic helix-loop-helix transcriptional factors involved in the muscle cell growth and differentiation (Chargé and Rudnicki, 2004). Previous studies have shown that TGF-β1 prevents muscle cell proliferation and differentiation by inhibiting the transcriptional activity of MyoD and myogenin (Vaidya et al., 1989; Martin et al., 1992). However, the molecular mechanism of how TGF-β1 affects the function of the myogenic regulatory factors is not yet well understood.

Transforming growth factor-β1 signaling pathway consists of 2 general steps that are required to carry the TGF-β1 signal to the target genes. Initially, TGF-β1 binds to membrane receptors, type I and type II, and assembles a receptor complex that phosphorylates Smad2/3 proteins. Subsequently, the activated Smad2/3 complexes are able to regulate transcription of the target genes (Mehra and Wrana, 2002). During myogenesis, Smad3, in response to TGF-β1, may interfere with the association of MyoD and the E-box, a conserved DNA sequence located in the regulatory region of many muscle-specific genes (Murre et al., 1989; Davis et al., 1990), and mediate the inhibition of myogenic differentiation (Liu et al., 2001). Smad7, one of the inhibitory Smads, reduces the signaling activity of Smad2/3 by inhibiting Smad2/3 phosphorylation (Nakao et al., 1997). However, the regulation of TGF-β1-induced Smad-mediated signaling pathway on skeletal muscle development remains unclear.

To respond to signals from the ECM including growth factors, cell adhesion receptors are also required during muscle growth and development. One of the means by which signals are transduced to the cells is through integrin receptors. Integrins are heterodimeric transmembrane cell adhesion receptors. Each integrin consists of {alpha}- and β-subunits that span the plasma membrane providing a transmembrane linkage for the bidirectional transmission of signal information between the ECM and cellular cytoskeletal network (Hynes, 1992). Beta 1 integrin, the most ubiquitous β subunit, associates with at least 12 different {alpha} subunits and forms the largest and most abundantly expressed subfamily playing a critical role in cell attachment, migration, proliferation, and differentiation (Berman et al., 2003). Beta 1 integrin has been shown to be involved in regulating muscle contraction through the interaction of the β1 integrin cytoplasmic domain with the cytoskeleton (Belkin et al., 1997), and is required for cell proliferation and survival (Velleman, 1999). During muscle development, β1 integrin subunit is critical in the adhesion of muscle cells (Neff et al., 1982; Decker et al., 1984) and myoblast fusion into multinucleated myotubes (Rosen et al., 1992). The {alpha}5β1 integrin is necessary for muscle differentiation (Boettiger et al., 1995) and prevents apoptosis (Zhang et al., 1995). If {alpha}5β1 integrin expression is reduced, the myoblasts will not properly adhere to the ECM and will likely undergo apoptosis. Transforming growth factor-β1 is a potent regulator of integrin-substrate interactions. For example, TGF-β1 results in an upregulation of several integrin subunits and a more adhesive phenotype (Ignotz and Massagué, 1987). However, the effect of TGF-β1 signaling on integrins during muscle formation, especially the β1 integrin subunit, is largely unknown.

The chicken Low Score Normal (LSN), genetic muscle weakness, was originally detected in 1977 at the University of Connecticut among F2 progeny in an out-cross of chickens with hereditary muscular dystrophy to a commercial White Leghorn stock. Low Score Normal birds have a loss in pectoralis major (p. major) muscle function and a decreased ability to right themselves when repeatedly placed on their backs (Velleman et al., 1993). Compared to normal birds, LSN birds have a 68% decrease in pectoral muscle mass and a 60% reduction in BW (Velleman et al., 1996). Velleman and Coy (1998) showed that LSN p. major muscle has elevated TGF-β1 expression levels. Because TGF-β1 is an inhibitor of both myoblast proliferation and differentiation (Allen and Boxhorn, 1987), the increased expression of TGF-β1 likely results in the decreased proliferation and differentiation of the LSN satellite cells, leading to the reduced muscle mass. Furthermore, both β1 integrin expression and localization are modified in LSN satellite cells compared to the control satellite cells (Velleman et al., 2000; Velleman and McFarland, 2004). The mechanism resulting in the differences in muscle growth and development between LSN and normal chickens remains to be elucidated. The present study used the LSN as a model to compare with normal muscle development to investigate how TGF-β1 affects in vivo muscle growth and development in both embryonic and posthatch stages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Birds

Specific-pathogen-free and LSN chickens used in the present study were from flocks maintained by the Ohio Agricultural Research and Development Center at The Ohio State University. The LSN phenotype was detected in 1977 at the University of Connecticut and reported by Velleman et al. (1993).

Injection of the Chicken Eggs

Fertile eggs from both the normal and LSN birds were injected with 100 ng of recombinant TGF-β1 (Pepro Tech Inc., Rocky Hill, NY) in a 100-µL volume of PBS or only PBS as a control at embryonic day (ED) 3 and incubated in a 37.5°C and 60% humidity egg incubator (NatureForm Hatchery Systems Inc., Jacksonville, FL). In brief, the egg shells were drilled with a Dremel tool resulting in a 1-mm-diameter hole, and then 100 ng of TGF-β1 in 100 µL of PBS or only PBS (control) was injected into the air sac area of the eggs at ED 3 using a 26-gauge 1/2 needle and 1-mL syringe as adapted from Zwilling (1959). One hundred fifty eggs from each chicken line were randomly split into 5 groups corresponding to respective sampling times: 60 eggs for ED 10, 30 eggs for ED 17, 20 eggs for 1 d posthatch, 20 eggs for 1 wk posthatch, and 20 eggs for 6 wk posthatch. Each group was randomly split in half for either the control or TGF-β1 treatment. The p. major muscle from both the control and TGF-β1-treated group was harvested at ED 10 and 17, and 1 d, 1 wk, and 6 wk posthatch. Body weight and p. major muscle were weighed and recorded at each sampling time.

Total RNA Extraction and cDNA Synthesis

The p. major muscle from each bird was removed, quick frozen in liquid nitrogen, and stored at –70°C until use. Total RNA was extracted from the tissue samples using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Reverse transcription (RT) of total RNA to a cDNA was conducted using Moloney murine leukemia virus reverse transcriptase (M-MLV; Promega, Madison, WI). In brief, an RNA-primer mix [1 µg of total RNA, 1 µL of 50 µM Oligo d(T)20, and nuclease-free water up to 13.5 µL] was incubated at 80°C for 3 min, followed immediately by incubation on ice. The reaction mix [5 µL of 5x First-Strand buffer, 1.25 µL of 10 mM deoxynucleoside triphosphate mix, 0.5 µL of RNasin (40 U/µL), 1 µL of M-MLV (200 U/µL), and nuclease-free water up to 11.5 µL] was added to the mixture. The complete reaction mixture was incubated at 55°C for 60 min, and then heated at 90°C for 10 min for inactivation.

Real-Time Quantitative PCR

Real-time quantitative PCR was performed using the DyNAmo Hot Start SYBR Green qPCR kit (Finnzymes, Woburn, MA). The PCR reaction consisted of 2 µL of the RT reaction mixture diluted with 25 µL of nuclease-free water, 10 µL of 2x master mix provided by the manufacturer, 250 nM of each of the forward and reverse primers, and nuclease-free water up to 20 µL. Reaction components were assembled in low-profile multiplates (Bio-Rad Laboratories Inc., Hercules, CA) and sealed with ThermalSeal RT (Phenix Research Products, Candler, NC). Primers used in the amplification of MyoD, myogenin, Smad3, Smad7, β1 integrin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed from published sequences as listed in Table 1Go. The specificity of the primers was confirmed by DNA sequence analysis of the amplified PCR product. The real-time PCR amplification was done in a DNA Engine Opticon 2 real-time system (MJ Research, Reno, NV). For all the amplified genes, the cycling program was initiated with a hot-start step at 95°C for 15 min. The cycling program for GAPDH and β1 integrin was performed as described in Li et al. (2006). The cycling conditions for MyoD and myogenin were denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 45 s for 34 cycles with a final elongation for 5 min at 72°C. The PCR cycling conditions for Smad3 and Smad7 were denaturation for at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s for 34 cycles with a final elongation at 72°C for 5 min. Standard curves were constructed for MyoD, myogenin, Smad3, Smad7, β1 integrin, and GAPDH with serial dilutions of the purified PCR products from each gene. The PCR products were purified by agarose gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). 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 MyoD, myogenin, Smad3, Smad7, and β1 integrin were normalized to GAPDH expression.


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  		Table 1. Primer sequences for real-time PCR
 
Immunohistochemistry

Pectoralis major muscle tissue was dissected from ED 10 and 17 embryos, and 1 d, 1 wk, and 6 wk post-hatch birds, and frozen by placing the tissue above liquid nitrogen permitting the liquid nitrogen vapor to freeze the tissue, and stored at –70°C until use. The muscle tissue samples were cut into 0.5-cm3 pieces and embedded in Tissue-Tek Optimal Cutting Temperature freezing media (Sakura Finetek, Torrance, CA) at –20°C. Cross-sections (7 µm) were cut using a Leica Model Cryostat (Leica, Nussloch, Germany), mounted on a Superfrost Plus slide (Fisher, Pittsburgh, PA), air-dried at least 1 h, and stored at –70°C.

The slides were removed from the –70°C storage, equilibrated to room temperature, and then fixed in ice-cold acetone for 5 min. The slides were washed with 1x Tris-buffered saline (20 mM Tris-HCl and 500 mM NaCl; pH 7.5; TBS) for 5 min. After washing, the sections were covered with 10% goat serum (Gibco BRL, Grand Island, NY) in 1x TBS with 1% BSA and incubated for 1 h at room temperature. At the end of the incubation, the goat serum was removed and the slides were then covered with 200 µL of β1 integrin cell substrate attachment monoclonal antibody (Developmental Studies Hybridoma Bank at the University of Iowa; 1:100 in 1x TBS containing 1% BSA; Lennon et al., 1991) at 4°C for 17 h. Negative control slides that did not include the β1 integrin primary antibody were also processed in the 1x TBS solution containing 1% BSA. The slides were then washed 2x for 10 min each in 1x TBS and once for 10 min in 1x TBS containing 0.5% BSA. After washing, the slides were incubated at room temperature for 2 h in a 1:200 dilution of goat anti-mouse rhodamine (Chemicon International Inc., Temecula, CA) in the 1x TBS containing 1% BSA and incubated at room temperature in the dark for 2 h. The slides were washed 2x for 5 min in 1x TBS and then mounted in aqueous mounting media GEL/ MOUNT (Biomeda, Foster City, CA) and covered with a coverslip (Fisher Scientific). The immunolocalization of β1 integrin was observed with an Olympus IX 70 microscope (Olympus America Inc., Melville, NY) and recorded with an Optronics digital camera (Olympus America Inc.).

Statistical Analysis

Statistical analysis was performed on the data using a 2-way ANOVA to estimate the effect of line, treatment, and their interaction on BW, p. major muscle weight, and mRNA expression at each sampling time. Differences were considered significant at the 0.05 level of probability.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of TGF-β1 on BW and P. Major Muscle Weight

During embryonic development, the BW of the normal embryos was not significantly different from that of the LSN embryos (Figure 1AGo). After hatch, the BW of the normal chickens was greater than that of the LSN from 1 d to 6 wk (Figure 1BGo). The TGF-β1-treated chickens did not differ in BW compared to the control during normal or LSN embryonic development and posthatch growth (Figure 1A and BGo). There was no interaction between line and treatment at any age for BW. The p. major muscle weight of LSN birds was significantly decreased compared to that of the normal from 1 d to 6 wk posthatch (Figures 1C and DGo). In normal birds, p. major muscle weight was significantly reduced by TGF-β1 at 1 wk posthatch compared to the control, but there was no difference for LSN resulting in an interaction between line and treatment. No difference was observed at other developmental ages. The LSN p. major muscle mass did not differ significantly between the control and TGF-β1-treated group.


Figure 1
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  		Figure 1. The effect of transforming growth factor-β1 (TGF-β1) on BW and pectoralis major (P. major) muscle weight during normal and Low Score Normal (LSN) development. Each egg was injected with 100 ng of TGF-β1 in 100 µL of PBS or only 100 µL of PBS at embryonic day (ED) 3. A) BW at 10 and 17 ED; B) BW at 1 d, 1 wk, and 6 wk posthatch; C) pectoralis major muscle weight at 17 ED, 1 d, and 1 wk posthatch; and D) pectoralis major muscle weight at 17 ED, 1 d, 1 wk, and 6 wk posthatch. Bars represent the standard of the mean. a–cWeights with no common letter within a sampling time were significantly different (P < 0.05).

 
Endogenous TGF-β1 Expression in P. Major Muscle

Endogenous TGF-β1 expression in LSN p. major muscle was increased from 17 ED to 6 wk posthatch and significantly induced by 30 and 29% at 17 ED and 6 wk posthatch, respectively, compared to the normal (Figure 2A and BGo). At 10 ED, there was a significant interaction between line and treatment because of a difference between the treatment in the normal but not in the LSN. In TGF-β1-treated p. major muscle, the expression of TGF-β1 was significantly decreased at 17 ED compared to the control without TGF-β1 treatment in both normal and LSN birds. The normal p. major muscle had greater expression of TGF-β1 at 6 wk posthatch, whereas the expression of TGF-β1 in TGF-β1-treated LSN p. major muscle did not differ from the control group except at 17 ED.


Figure 2
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  		Figure 2. Endogenous transforming growth factor-β1 (TGF-β1) mRNA expression in TGF-β1-treated and the control birds during normal and Low Score Normal (LSN) chicken growth and development. Each egg was injected with 100 ng of TGF-β1 in 100 µL of PBS or only 100 µL of PBS at embryonic day (ED) 3. A) TGF-β1 expression at 10 and 17 ED; and B) TGF-β1 expression at 1 d, 1 wk, and 6 wk posthatch. a–cValues with no common letter within a sampling time were significantly different (P < 0.05). Bars represent the standard error of the mean.

 
Effect of TGF-β1 on the Expression of Smad3, Smad7, MyoD, and Myogenin

Normal p. major muscle had greater expression of Smad3 than LSN at both 10 ED and 1 wk posthatch (Figure 3A and BGo). The expression of Smad3 in normal p. major muscle was significantly reduced by TGF-β1 at 10 ED, 1 d, and 1 wk posthatch. The expression of Smad7 in LSN p. major muscle was significantly lower than normal muscle at 17 ED and 6 wk posthatch (Figure 3C and DGo). At 10 ED and 1 wk, the interaction between line and treatment was significant. After hatch, the expression of Smad7 was significantly reduced by TGF-β1 in both normal and LSN p. major muscle at 1 d and 6 wk posthatch. However, the TGF-β1-treated LSN p. major muscle did not significantly differ in Smad3 expression compared to the control during both embryonic and posthatch development. The TGF-β1-treated p. major muscle did not have any significant difference in Smad7 expression, compared to the control during either normal or LSN embryonic development.


Figure 3
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  		Figure 3. The effect of transforming growth factor-β1 (TGF-β1) on Smad3 and Smad7 mRNA expression during normal and Low Score Normal (LSN) development. Each egg was injected with 100 ng of TGF-β1 in 100 µL of PBS or only 100 µL of PBS at embryonic day (ED) 3. A) Smad3 expression at 10 and 17 ED; B) Smad3 expression at 1 d, 1 wk, and 6 wk posthatch; C) Smad7 expression at 10 and 17 ED; and D) Smad7 expression at 1 d, 1 wk, and 6 wk posthatch. a–cValues with no common letter within a sampling time were significantly different (P < 0.05). Bars represent the standard error of the mean.

 
Compared to normal p. major muscle, MyoD expression in LSN p. major muscle was significantly decreased at 17 ED and then increased at 1 wk and 6 wk posthatch (Figure 4A and BGo). During embryonic development, the expression of MyoD was significantly decreased from 35 to 48% by the injection of exogenous TGF-β1 from 10 to 17 ED in normal p. major muscle but the interaction of line and treatment was significant at 17 ED (Figure 4AGo). At 1 d posthatch, the expression of MyoD was significantly decreased 52% by TGF-β1 in LSN p. major muscle. From 1 d to 6 wk posthatch, the TGF-β1-treated normal p. major muscle did not have significant difference in MyoD expression, compared to the control (Figure 4BGo). The LSN p. major muscle did not differ significantly in MyoD expression between the TGF-β1-treated and control groups during either embryonic development (Figure 4AGo) or 1 wk and 6 wk posthatch (Figure 4BGo). The expression of myogenin in LSN p. major was significantly increased compared to normal muscle from 1 d to 1 wk posthatch, but did not differ during embryonic development. In the TGF-β1-treated normal p. major muscle, the expression of myogenin was significantly decreased by 29% at 1 wk posthatch. After hatch, myogenin expression in the TGF-β1-treated LSN p. major muscle was significantly reduced 43 and 32% at 1 d and 1 wk posthatch, respectively (Figure 4DGo). In the TGF-β1-treated normal p. major muscle, the expression of myogenin was not significantly different compared to the control during the embryonic stage and 1 d and 6 wk posthatch growth, resulting in a significant interaction between line and treatment after hatch. Similar to normal p. major muscle, myogenin expression in LSN p. major muscle treated with TGF-β1 did not differ from the control during embryonic development (Figure 4C and DGo).


Figure 4
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  		Figure 4. The effect of transforming growth factor-β1 (TGF-β1) on MyoD and myogenin mRNA expression during normal and Low Score Normal (LSN) development. Each egg was injected with 100 ng of TGF-β1 in 100 µL of PBS or only 100 µL of PBS at embryonic day (ED) 3. A) MyoD expression at 10 and 17 ED; B) MyoD expression at 1 d, 1 wk, and 6 wk posthatch; C) myogenin expression at 10 and 17 ED; and D) myogenin expression at 1 d, 1 wk, and 6 wk posthatch. a–cValues with no common letter within a sampling time were significantly different (P < 0.05). Bars represent the standard error of the mean.

 
Effect of TGF-β1 on the Expression and Localization of β1 Integrin

The expression of β1 integrin in normal p. major muscle was significantly greater than that in the LSN during both embryonic and posthatch development except for 1 wk posthatch (Figure 5A and BGo). In normal p. major muscle, the expression of β1 integrin was significantly reduced by TGF-β1 from embryonic through posthatch development. However, β1 integrin expression in LSN p. major muscle was significantly increased by TGF-β1 treatment at 10 and 17 ED, and did not differ after hatch compared to the control. There was a significant interaction of line and treatment at 10 and 17 ED and at 1 wk posthatch.


Figure 5
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  		Figure 5. The effect of transforming growth factor-β1 (TGF-β1) on β1 integrin mRNA expression during normal and Low Score Normal (LSN) chicken growth and development. Each egg was injected with 100 ng of TGF-β1 in 100 µL of PBS or only 100 µL of PBS at embryonic day (ED) 3. A) β1 integrin expression at 10 and 17 ED; and B) β1 integrin expression at 1 d, 1 wk, and 6 wk posthatch. a–cValues with no common letter within a sampling time were significantly different (P < 0.05). Bars represent the standard error of the mean.

 
During embryonic and posthatch development, the localization of β1 integrin was observed in normal and LSN p. major muscle. At 17 ED, β1 integrin was localized in the muscle fiber membrane and cell-cell contact areas in both the control and TGF-β1-treated p. major muscle (Figure 6Go). No significant change in localization was observed in the TGF-β1-treated normal and LSN p. major muscle compared to the control. Similar to 17 ED, at 1 d posthatch, β1 integrin was clustered at the edge of the cells or capillary wall in both normal and LSN p. major muscle (Figure 7Go). No significant difference in β1 integrin localization was observed between the control and TGF-β1-treated birds at any other ages.


Figure 6
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  		Figure 6. The localization of β1 integrin in transforming growth factor-β1 (TGF-β1)-treated normal and Low Score Normal (LSN) chickens. A) Pectoralis major muscle from the control normal chicken at 17 embryonic day (ED); B) pectoralis major muscle from the TGF-β1-treated normal chicken at 17 ED; C) pectoralis major muscle from the control LSN chicken at 17 ED; and D) pectoralis major muscle from the TGF-β1-treated LSN chicken at 17 ED. The tissue samples were immunostained with β1 integrin cell substrate attachment monoclonal antibody; the arrows indicate the localization of β1 integrin, and scale bar = 20 µm.

 

Figure 7
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  		Figure 7. The β1 integrin localization in transforming growth factor-β1 (TGF-β1)-treated normal and Low Score Normal (LSN) chickens. A) Pectoralis major muscle from the control normal chicken at 1 d posthatch; B) pectoralis major muscle from the TGF-β1–treated normal chicken at 1 d posthatch; C) pectoralis major muscle from the control LSN chicken at 1 d posthatch; and D) pectoralis major muscle from the TGF-β1–treated LSN chicken at 1 d posthatch. The tissue samples were immunostained with β1 integrin cell substrate attachment monoclonal antibody; the arrows indicate the localization of β1 integrin, and scale bar = 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the effect of TGF-β1 on skeletal muscle growth and development was examined during both normal and LSN embryonic development and posthatch growth. The weight of LSN p. major muscle was significantly decreased from 1 d to 6 wk posthatch, compared to normal p. major muscle. Velleman et al. (1996) demonstrated that the weight of the p. major muscle expressed as a percentage of BW was 3.2% in the normal birds and 1.9% in the LSN birds at 1 wk posthatch. Endogenous TGF-β1 expression was increased in the LSN p. major muscle at both 17 ED and 6 wk posthatch, compared to the normal. A previous study showed that the expression of TGF-β1 was increased in the LSN birds compared to the normal birds at 1 d posthatch using slot-blot analysis (Velleman and Coy, 1998). The reduction in LSN p. major muscle weight may be associated with the increased expression of endogenous TGF-β1. After treating with exogenous TGF-β1, normal p. major muscle weight was significantly decreased compared to the control at 1 wk posthatch, although BW was not significantly affected by TGF-β1. Transforming growth factor-β1 is a potent inhibitor of myoblast growth and differentiation (Allen and Boxhorn, 1987). The reduction in the p. major muscle weight is likely due to TGF-β1-induced inhibition of muscle cell proliferation and differentiation during embryonic development and posthatch growth. To test this, myogenic regulatory factor expression was examined as well as the Smad3-mediated TGF-β1 signaling pathway.

The TGF-β1-treated normal p. major muscle had a significant decrease in Smad3 expression at 10 ED, and 1 d and 1 wk posthatch. The expression of Smad3 in normal p. major muscle was significantly greater compared to LSN muscle at 10 ED and 1 wk posthatch. The increased TGF-β1 expression in LSN p. major muscle may reduce the expression of Smad3 in LSN p. major muscle compared to normal muscle. The expression of Smad7 was not significantly affected by TGF-β1 during embryonic growth in either normal or LSN birds. In an in vitro study, Li et al. (2008) showed that TGF-β1 reduced Smad3 expression in chicken satellite cells, but had no effect on Smad7 expression. The TGF-β1 signal was carried by Smad3 into the nucleus, leading to the inhibition or activation of the target gene transcription. Liu et al. (2001) showed that a Smad3-mediated TGF-β1 signaling pathway regulates muscle cell proliferation and differentiation by affecting the activity of myogenic regulatory factors. The reduction in Smad3 expression by TGF-β1 may indicate a negative regulatory feedback to TGF-β1-induced signaling pathway during skeletal muscle growth and development.

The myogenic regulatory factors MyoD and myogenin have been shown to be targeted by TGF-β1 signaling (Vaidya et al., 1989; Martin et al., 1992; Liu et al., 2001). During embryonic development, MyoD expression in normal p. major muscle was significantly reduced by TGF-β1. Myogenin expression was decreased by TGF-β1 treatment at 1 wk posthatch in normal p. major muscle. During myogenesis, MyoD is expressed in proliferating myoblasts and then myogenin is upregulated, leading to terminal differentiation (Molkentin and Olson, 1996). The reduction of MyoD expression in response to TGF-β1 may cause myoblast proliferation to arrest during embryonic development. A decrease in myogenin expression during the posthatch period may result in a reduction in myoblast differentiation. The reduction of both MyoD and myogenin is likely correlated with the decreased p. major muscle growth observed in the TGF-β1-treated normal birds. However, LSN skeletal muscle cells had a different cellular response to TGF-β1 from the normal cells. Compared to normal p. major muscle, TGF-β1-treated LSN p. major muscle had a delayed decrease in MyoD expression at 1 d posthatch in response to TGF-β1, compared to the normal birds, which had a reduction in MyoD expression at 17 ED. Myogenin expression was reduced by TGF-β1 in the LSN p. major muscle at 1 d and 1 wk posthatch. These data indicated that the reduction of both MyoD and myogenin expression by TGF-β1 was likely associated with LSN muscle cell proliferation and differentiation.

The communication between myoblasts and the ECM during skeletal muscle growth and development is mediated by integrin cell adhesion receptors. The data from the present study showed that the expression of β1 integrin in normal p. major muscle was dramatically reduced by TGF-β1 from embryonic to posthatch growth. An in vitro study showed that the addition of TGF-β1 can inhibit β1 integrin expression in normal satellite cells (Li et al., 2006). Thus, the reduction of β1 integrin is likely associated with the decreased p. major muscle weight in the TGF-β1-treated normal chicken. The expression of β1 integrin in normal p. major muscle was significantly greater compared to LSN through embryonic development and posthatch growth. Velleman et al. (2000) showed that β1 integrin protein levels were lower in the LSN compared to normal p. major muscle. In LSN satellite cell cultures, β1 integrin was not associated with the cell-to-cell contact area, and the LSN satellite cells underwent apoptosis (Velleman and McFarland, 2004). The muscle cells required appropriate expression and localization of β1 integrin during cell proliferation and differentiation, allowing for proper muscle cell migration and muscle formation. Loss of cell integrin attachment to the substrate results in muscle cell apoptosis (Zhang et al., 1995). The decreased β1 integrin expression in LSN p. major muscle is likely due to myoblast response to the increased TGF-β1 level in LSN birds, which is associated with decreased muscle growth and altered muscle structure observed in LSN birds.

The role of TGF-β1 on myogenesis has been well documented in in vitro studies. However, only recently has an in vivo effect for TGF-β1 begun to be elucidated. The present study demonstrates that TGF-β1 reduces both skeletal muscle cell proliferation and differentiation by inhibiting MyoD and myogenin expression through a Smad3-mediated TGF-β1 mediated signaling pathway. The reduction in β1 integrin expression in response to TGF-β1 may also contribute to the effect of TGF-β1 on muscle growth and development.

Myostatin, a member of the TGF-β family recognized as an inhibitor of myoblast proliferation and differentiation, has drawn more attention in animal agriculture. The mutation of myostatin gene results in a "double muscling" phenotype in Belgian Blue and Piedmontese cattle (McPherron and Lee, 1997). However, little is understood about the regulation of growth factors as it relates to skeletal muscle growth and development. To obtain maximal muscle growth and maintain muscle quality in as short a period of time as possible is a goal to maintain low production costs in the poultry industry. Thus, understanding the mechanisms involved in the regulation of muscle growth and development is critical for increasing the growth rate and muscle mass, and maintaining a high-quality product. The findings from the current study will lead to a better understanding of mechanisms underlying the signaling events mediated by TGF-β1 with regard to muscle development and growth. Further studies are needed to elucidate how a TGF-β1-involved mechanism manipulates the growth and development of skeletal muscle during different developmental stages.


   FOOTNOTES
 
1 Salary and research support to S.G.V. were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and the Midwest Poultry Consortium. Back

Received for publication June 10, 2008. Accepted for publication October 2, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Effect of transforming growth factor-{beta}1 on decorin expression and muscle morphology during chicken embryonic and posthatch growth and development
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