Muscle growth and development from the embryonic to the adult stage of an organism consists of a series of exquisitely regulated and orchestrated changes in expression of genes leading to muscle maturation. Genetic selection for body weight and muscle mass is ultimately based on differential expression of genes, and this approach has resulted in dramatic improvements in turkey growth rate, breast muscle mass, and feed efficiency. However, selection for these performance traits has inadvertently led to other undesirable traits including myopathies [26, 27], cardiomyopathies [28, 29], skeletal deficiencies [11, 30], and meat quality defects such as the pale, soft, and exudative (PSE) condition [31, 32] and associated decreased meat protein functionality . The goals in the current study were to exploit the capability of a microarray approach to identify functional pathways of genes and thus characterize molecular events associated with muscle development and between genetic lines of turkeys with different growth rates.
Obvious phenotypic differences exist between RBC2 birds and those from F or commercial lines. The F line turkeys gain body weight faster and yield significantly heavier breast muscles than the RBC2 birds . In addition, satellite cells from the F line have faster proliferation and differentiation rates than those from the RBC2 line . Further, muscle damage with age is significant between lines as birds from the F line display a greater degree of muscle fiber fragmentation and areas of hypercontraction compared to those of the RBC2 . These phenotypic differences are likely the results of differential gene expression throughout development.
This study identified over 3000 genes affected by developmental stage (Experiment 1), but only 16 genes were identified as being differentially expressed by overall effect of genetic line (Experiment 2). When considering effect of line within each developmental stage, the highest number of differences occurred early in development (18de stage). Expression changes in myogenic regulatory factor genes have previously been observed between the F and RBC2 lines at the embryonic stage , and satellite cells from F birds have been shown to have increased proliferation rates relative to RBC2 birds in vitro . These results agree with the microarray observations and suggest that phenotypic differences between the two lines may largely be determined embryonically during myogenesis. It is possible that subtle differences in gene expression observed in the current study may be responsible for large phenotypic differences in body and breast muscle weight , breast muscle morphology , and meat quality characteristics  between the RBC2 and F lines at market age.
The Ingenuity Pathway Analysis (IPA) enabled the clustering of differentially expressed genes into functional categories, some of which were expected, while others were novel. Expected differences provided biological validation of the microarray approach, while the novel differences provided new paths to pursue in furthering our understanding of differences in turkey skeletal muscle growth and development. Many genes that may act as master regulators during development appeared in more than one Network, Function, or Canonical Pathway. The identified gene expression differences were grouped by function and discussed, highlighting several individual genes of interest within each group.
Extracellular Matrix Genes
Previous studies have demonstrated that the ECM comprising the connective tissue surrounding muscle can interact with growth factors, regulate cellular signal transduction pathways, and affect growing and developing muscle fibers [34–36]. Genes that fell into numerous functions or canonical pathways as well as some genes that were not included in IPA networks were selected for this group. The ECM is composed mainly of collagens and non-fibrous glycoproteins like proteoglycans. The expression of three proteoglycans: versican, glypican-1, and syndecan-4, was quantified in this study. Membrane-associated proteoglycans in the ECM are known to regulate numerous growth factors, the major constituents that regulate activation, proliferation, and differentiation of satellite cells . A predominant example is FGF2, a potent stimulator of muscle fiber proliferation and a strong inhibitor of differentiation [35, 37]. This function would explain the high FGF2 expression at 18de and 1d in the current study, as these are periods of myoblast or satellite cell proliferation. The interaction between FGF2 and the heparan sulfate proteoglycans glypican-1 and syndecan-4 is required for high-affinity binding of FGF2 to its cellular receptor . Differences in expression of syndecan-4 have been observed in vitro between developing RBC2 and F line satellite cells , and RBC2 satellite cell expression of syndecan-4 as well as glypican-1 were altered with the addition of FGF2 . Satellite cells from syndecan-4 knockout mice display defective patterns of activation, proliferation, and differentiation , indicating a crucial role in muscle development that is also supported for turkeys in the current study. In addition, syndecan-4 and FGF2 appear to have similar expression profiles, especially at 1d. Versican is a chondroitin sulfate proteoglycan that was expressed predominantly at the 18de stage in this study. Fernandez et al. (1991) hypothesized that this protein's high embryonic expression at the time of fiber formation may be involved in fiber spacing, establishing the morphological structure of the muscle in chicks . The space between muscle fibers may play a critical role in the water-holding capacity of mature muscle and may therefore be associated with quality defects like PSE meat. It is therefore possible that characteristics that contribute to meat quality are decided very early in myogenesis.
Dominant negative mutations in the COL6A1 gene, the only collagen that was investigated in the current study, are known to lead to Bethlem myopathy  and Ulrich congenital muscle dystrophy ; COL6A1 knockout mice exhibit sarcolemmal disorganization . Taken together, it is clear that expression of genes whose proteins are located in the ECM of skeletal muscle likely contribute significantly to myogenesis in RBC2 and F turkeys.
Cell Death/Apoptosis Genes
Cell death and apoptosis are necessary events in muscle differentiation and maintenance. In addition, other reports have shown similarities between mechanisms of apoptosis and myoblast differentiation, suggesting that similar pathways and effectors may be in use [44, 45]. Therefore, it was logical to further evaluate the expression of several differentially expressed genes associated with these functions. For example, caspase-3, an "executioner" cysteine protease in apoptosis , is necessary for proper differentiation of C2C12 myoblasts . However, several reports have shown that this role is separate from apoptosis [44, 45] although it employs similar mechanisms, such as DNA strand breakage .
A component of the microfilament, GAS2, is a substrate for caspase-3 and is involved in microfilament reorganization, possibly an early event in apoptosis . The protein is expressed in embryonic mouse limbs and involved in apoptosis during interdigital development; GAS2 may also be involved in myogenesis, possibly in the fusion of myoblasts to form myotubules . In addition, GAS2 can regulate p53 action and apoptosis through its inhibition of calpain . Nestin is the initial intermediate filament protein expressed during myogenesis [52–54], and phosphorylation of this protein is associated with the disassembly of intermediate filaments during mitosis . Nestin is also associated with increased survival of rat vascular smooth muscle cells under stress . Our results are in agreement as nestin is expressed predominantly at the 18de stage in turkey skeletal muscle when myoblasts are proliferating.
Another intriguing gene was DAP, which was expressed most highly at the 18de stage and decreased throughout development for both RBC2 and F birds. The human homologue of the gene was first identified in surviving HeLa cells treated with an antisense cDNA library and the cytokine interferon-γ to induce apoptosis . Very little is known about this gene other than it seemed to be a candidate for positive mediators of cell death [56, 57] and that it may regulate autophagy in injured planarians  and in amino acid-starved HeLa cells . In addition, DAP may play a role as a regulator of turkey skeletal muscle proliferation and differentiation as shown by our group's recent work with satellite cells in vitro (Vellemen et al., manuscript in preparation).
Ca2+ Signaling/Muscle Function Genes
The Ca2+ ion acts as a second messenger in cell signaling and in skeletal muscle contraction. Aberrant Ca2+ signaling postmortem likely plays an important role in the occurrence of meat quality defects that can lead to significant economic losses in the pork and poultry industries. During the postmortem conversion of muscle to meat, increased Ca2+ release from the sarcoplasmic reticulum while the muscle temperature is still high is thought to be responsible for skeletal muscle hypermetabolism that accelerates pH decline, leading to protein denaturation and loss of protein functionality [60, 61]. A point mutation in the "halothane gene" or ryanodine receptor 1 (RYR1), the skeletal muscle sarcoplasmic reticulum Ca2+ release channel, has been identified in the pig  and has been associated with porcine stress syndrome (PSS), a sometimes fatal condition of malignant hyperthermia that is triggered by stress and is a major contributor to the development of PSE pork . Genetic selection by pig breeders away from this mutation has not fully resolved the PSE problem. However, RYR1 became a logical candidate for examination in the turkey, a species that has been similarly selected for rapid lean muscle growth and also exhibits meat quality defects such as PSE. Further investigation of this protein in the turkey revealed a higher affinity for ryanodine in heavier commercial turkeys than those from the RBC2 line , suggesting a higher open-state probability of the Ca2+ release channel. Multiple alternatively spliced products were later discovered in the avian skeletal muscle ryanodine receptor isoform, αRYR, in RBC2 and commercial turkeys , and these alternative splicing sites are clustered in regions of the gene associated with increased frequency of mutations . Recently, alternative splicing and changes in αRYR expression have also been implicated in the occurrence of PSE meat in broiler chickens .
Several genes involved in Ca2+ metabolism were identified as differentially expressed during development in this study. Calreticulin functions as a Ca2+ -binding chaperone and aids in maintaining Ca2+ homeostasis in the ER lumen of cells . This protein also appears to be mainly expressed early in murine cardiac development and is essential for proper cardiac development . Calmodulin is another Ca2+ -binding protein that sensitizes RYR1 to activation at nanomolar concentrations of Ca2+ and inhibits RYR1 at micromolar concentrations . However, more recent work suggests that calmodulin is not an essential regulator of RYR1 . Nevertheless, calmodulin plays a central role in calcium signal transduction and the developmentally related changes in gene expression suggest a key role for this protein.
Myostatin is a highly-conserved transforming growth factor-β (TGF-β) family member that strongly inhibits both hyperplasia and hypertrophy. Mutations or knockout of the gene result in an extreme increase in skeletal muscle mass in cattle and in mice . In the current study, a dramatic decrease of myostatin expression was observed at 1d post-hatch, which agrees with our understanding of myostatin function as myofibers are rapidly undergoing hypertrophy at this stage. In addition, line differences at 18de and 16wk were observed, with RBC2 birds expressing myostatin significantly higher than F birds at 18de. This result could imply that F birds are able to undergo a higher degree of hyperplasia and actually produce more mature muscle fibers, leading to greater muscle mass. At 16wk, F-line birds expressed significantly more myostatin, suggesting that the faster growth rate of the heavier birds plateaus sooner than the slower-growing RBC2-line birds. Activin receptor type IIB (ActIIBR) functions as a serine/threonine kinase receptor for myostatin as well as other TGF- β family members, and binding of these ligands to ActIIBR activates the Smad signal transduction pathway to regulate gene expression . Inhibition of myostatin activity by construction of a dominant-negative ActRIIB lacking a kinase domain resulted in increased muscle hyperplasia and hypertrophy in mice, as well as competition with the TGF-β family member follistatin . Treatment of chicken fetal myoblasts with myostatin altered expression of genes involved in myogenic differentiation, cell architecture, energy metabolism, signal transduction and apoptosis , suggesting that increased myostatin expression in the current study could lead to similar changes.
Troponins are key proteins that are regulated by changes in intracellular Ca2+ concentrations and are responsible for striated muscle contraction. The three subunits of troponin interact with each other to bind Ca2+ (TNNC), inhibit myosin ATPase activity (TNNI), and to bind tropomyosin (TNNT) . Transcript abundance of the fast/skeletal muscle isoform of TNNT, TNNT3, increased with development in the current study, peaking at 16wk. Interestingly, the cardiac isoform, TNNT2, was chiefly expressed in the embryonic skeletal muscle, and its expression decreased during skeletal muscle development, which is consistent with previous findings in chicken [75, 76]. Expression of another slow-twitch muscle/cardiac isoform of troponin, TNNI1, followed this pattern as well. The expression of all of the troponin isoforms is also in agreement with what is known about myofibrillar protein expression in rat skeletal muscle, as slow/cardiac forms are expressed during the development of myofibrils with a switch to fast/skeletal forms during postnatal growth and muscle regeneration . These genes involved in muscle growth and Ca2+ signaling may be valuable candidates for changes observed in turkey skeletal muscle development.
Miscellaneous/Genes of Unknown Function
One of the goals of the current study was to identify novel genes with unknown or uncertain functions that may play important roles in skeletal muscle development. These genes may play important roles in myogenesis and development even though their annotations and functions have yet to be defined or may not have clear roles in skeletal muscle. In the current study, one gene without known annotation but identified as very highly differentially expressed by microarray analysis was chosen for further confirmation by qPCR along with others that did not easily fit into a functional category. These genes all showed line differences at 1d, indicating a possible important role during muscle hypertrophy that may help explain phenotypic differences between the slow-growing randombred line and the line selected for increased 16-wk body weight.
Several genes identified in this study were first discovered and their activities characterized in tissue types other than skeletal muscle. For example, spondin 2, (also known as mindin), which fell into the ECM group in our study, is expressed in spleen, lymph nodes, and dendritic cells and may be crucial to immune function . In the current study, spondin 2 was expressed at highest levels at 1d post-hatch, over 11 times higher than its 18de expression in both turkey lines. The MGP gene was first cloned in chickens and found in highest levels in bones and relatively low levels in skeletal muscle . Previous studies indicate its role in the inhibition of vascular calcification, as MGP knockout mice die within 2 months due to blood vessel rupture . It may also be important in intracellular calcium homeostasis . While MGP was grouped into the "Cell Death" function by the IPA software, its association with apoptosis is not clear . It was another gene that appeared to be "turned on" at 1d post-hatch, with 80.28 and 55.38 times the 18de expression in RBC2 and F lines, respectively. Thus, this study has begun to uncover novel roles of these genes, which have been characterized in other tissues, in muscle growth and development.
Other interesting findings include the variation in the timing and extent of expression during development. A few stand-out genes were highly expressed at only one stage of development, clearly turned on or off in what appears to be carefully choreographed regulation. During the embryonic stage, when muscle cell hyperplasia is occurring, versican, glypican-1, betaparvin, nestin, TNNI1, and TNNT2 were expressed at least 5 times, and in the case of nestin greater than 100 times, higher than later developmental stages. Spondin 2 and MGP were both highly expressed at 1d posthatch with very little expression at 18de or 16wk. The NT5C3 and TNNT3 genes peaked at 16wk of development.