Long-term selection of chickens for body weight alters muscle satellite cell behaviors

Long-term selection of chickens for body weight alters muscle satellite cell behaviors A. E. Geiger, M. R. Daughtry, C. M. Gow, P. B. Siegel, H. Shi, and D. E. Gerrard1 Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, 24061 muscle fibers from HWS chickens possessed more SC than those from LWS. Further analysis showed that the SC pool from HWS muscles contained a higher percentage of activated SC compared to that of LWS. When isolated SC from HWS and LWS muscles were cultured, HWS SC exhibited greater abilities to proliferate and differentiate than those SC from LWS. To test whether the observed in vitro differences in SC properties could be confirmed in vivo, we subjected chicken breast muscle to barium chloride to induce muscle injury and regeneration. Consistent with in vitro data, breast muscle in HWS chicken experienced a faster and more robust recovery than that of LWS, as evidenced by quicker regeneration and larger muscle fiber size. Taken together, these findings suggest divergent selection for body weight not only results in correlated responses in SC number, but also changes SC growth kinetics. Further dissection of the molecular mechanism will aid the identification of the target molecules for growth intervention in chickens.

ABSTRACT Muscle satellite cells (SC) are resident stem-like cells that play an integral role in skeletal muscle growth and repair. Understanding how SC maintain their identities and dynamic properties is critical to animal growth. However, the genetic and environmental factors governing SC behaviors and the underpinning mechanisms remain unknown. To explore whether genetic selection influences SC behaviors, we used 2 lines of chickens selected for over 50 generations with over a 10-fold difference in body weight at 56 d of age—the Virginia high weight selection (HWS) and low weight selection (LWS) lines. To study these 2 lines, we performed both in vivo and in vitro experiments. In vivo, we studied the abundance of SC in normal physiological settings and tested their functional roles in muscle regeneration using a muscle injury model. In vitro, we isolated SC from chicken skeletal muscle and assayed their ability to proliferate and differentiate under cultured conditions. Immunohistochemical staining of breast muscle (pectoralis major) revealed that

Key words: selection, satellite cell, muscle injury, regeneration 2018 Poultry Science 0:1–11 http://dx.doi.org/10.3382/ps/pey050

INTRODUCTION

tween the sarcolemma and the basal lamina (Muir et al., 1965). This close association with the fiber together with the surrounding microenvironment, including adjacent vasculature and other cell types in the extracellular matrix, provides unique niches within the tissues that help govern SC fate decisions (Bi and Kuang, 2012; Yin et al., 2013). In addition to supplying myonuclei to fibers during post-hatch muscle hypertrophy, SC also function to repair muscle tissue after damage in a process referred to as regenerative myogenesis. In adult muscles, SC remain in a state of quiescence until activation in response to various stimuli, such as growth factors, hormones, stretching, or other physical perturbations (Allen and Rankin, 1990; Bischoff, 1990). Once activated, SC orchestrate muscle regeneration by re-entering the cell cycle to increase numbers (proliferate), then subsequently exit the cell cycle to differentiate into multinucleated nascent myofibers, fuse to existing myofibers, or selfrenew and revert back to the quiescent state (Dumont

Adult skeletal muscle is composed of multinucleated muscle fibers whose primary function is to facilitate ambulation (Dumont et al., 2015). After hatch, muscle fiber numbers do not increase appreciably. Thus, post-hatch muscle growth in chickens is predominantly driven by myofiber hypertrophy, characterized by an increase in muscle fiber volume, namely, length and width (White et al., 2010; Rehfeldt et al., 2011). This growth, or hypertrophy, at the cellular level is thought to require continuous addition of nuclei to the muscle cell from a type of resident myogenic stem cell known collectively as satellite cells (SC) (Beauchamp, 2000). These muscle precursor cells were first discovered in 1961 by Alexander Mauro (Mauro, 1961) and are positioned be C 2018 Poultry Science Association Inc. Received August 21, 2017. Accepted March 28, 2018. 1 Corresponding author: [email protected]

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et al., 2015). From a molecular standpoint, SC status (quiescent vs. activated) can be tracked in muscles through the expression of muscle-specific transcription factors (Rehfeldt et al., 2011). Paired box 7 (Pax7) transcription factor, of particular significance, is expressed in the majority of quiescent SC and is routinely used as a marker of SC nuclei and restricted to a muscle cell lineage in skeletal muscle (Seale et al., 2000). In contrast, those cells expressing myogenic regulatory factors (MRF), such as MyoD, Myf5, MRF4, and myogenin, are indeed considered activated and ready to proceed with the myogenic program, depending on the nutrient cues (Halevy et al., 2004; Bentzinger et al., 2012). Divergent selection for body weight (BW) in poultry serves as an excellent tool to study the biological processes underlying a number of physical traits, including tissue growth, development, and maturation (Velleman et al., 2000; Johansson et al., 2010). At Virginia Tech, a long-term selection experiment based on 56 d BW using White Plymouth Rock chickens has resulted in 2 distinctly different lines: high weight selection (HWS) and low weight selection (LWS) (Siegel, 1962; Marquez et al., 2010). This selection experiment, continued for over 50 generations, has resulted in more than a 10-fold difference in BW between the lines (Dunnington et al., 2013; Yin et al., 2014) resulting with a 2fold difference in standard deviation units in each line (Jambui et al., 2017). Studies using these lines have focused on myriad characteristics resulting from selection on BW and include: feed consumption, metabolic and reproductive traits, muscle fiber type characteristics, and muscle gene expression profiles (Dunnington and Siegel, 1996; Yin et al., 2014). Not surprisingly, HWS birds grow faster and consume more feed, yet are more efficient than LWS birds (Dunnington and Siegel, 1996). Nonetheless, however, little is known about how this selection paradigm alters SC (muscle stem cells) behavior in the muscle. Hence, the aim of this research was to define and compare the disparities in muscle tissue growth between these 2 lines using in vitro and in vivo studies.

MATERIALS AND METHODS Chickens Chickens used in this study were males randomly selected from lines divergently selected for 58 generations for high (HWS) or low (LWS) 56 d BW. The foundation stock was White Plymouth Rock (Siegel, 1962) with selection procedures, husbandry, and population structure previously described (Marquez et al., 2010; Dunnington et al., 2013). Ages of chickens used herein were either 5, 56, or 91 d post hatch. Data collected at each age were used to address questions arising from initial studies on 56 d post-hatch chicken muscle. All animal procedures were approved

by the Virginia Tech Institutional Animal Care and Use Committee.

Tissue Sample Collection Individuals were euthanized by carbon dioxide (CO2 ) administration followed by cervical dislocation and sections of the pectoralis major (PM) and biceps femoris (BF) muscles were collected, placed in freezing compound (O.C.T. Thermo Fisher Scientific, Fisher Healthcare, Houston, TX), and immediately frozen in isopentane pre-cooled in liquid nitrogen. Samples were stored at –80◦ C until analyses. 10-micron thick sections were made using a Microm HM550 cyrostat (Thermo Fisher Scientific, Waltham, MA) that were then mounted on 3-aminoproprytriethoxysilanesy (silane, Sigma-Aldrich, St. Louis, MO) coated microscope slides and used for immunocytochemistry or hematoxylin and eosin staining.

Satellite Cell Isolation and Culture Muscle samples collected from the PM were briefly rinsed in 70% ethanol and transferred to sterile phosphate-buffered saline (PBS). The outermost 10 mm layer of the muscle was removed and discarded, and the remaining tissue was finely minced. Minced muscles were digested in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Gibco, Gaithersburg, MD) containing high glucose, Lglutamine, 110 mg/mL sodium pyruvate, 110 mg/mL sodium pyruvate, containing 0.8 mg/mL Pronase (Sigma-Aldrich, St. Louis, MO), and 1% penicillinstreptomycin (pen/strep, Sigma-Aldrich, St. Louis, MO) for 60 min at 37◦ C under 80 rpm agitation. Digestions were centrifuged for 6 min at 300 X g. Supernatants were discarded, and pellets were washed in PBS and re-centrifuged. Supernatants were discarded, and pellets were suspended in high-glucose DMEM with 1% pen/step. Pellets were triturated at least 15 times using a 10 mL serological pipette and re-centrifuged. Supernatants were collected and filtered through 40μm sterile filters. The flow-through was diluted with PBS and centrifuged. Supernatants were discarded, and cells were suspended in 5 mL of high-glucose DMEM containing 10% chicken serum (Gemini Bio-Product, West Sacramento, CA), 5% horse serum (American Type Culture Collection ATCC, Manassas, VA), 1% pen/strep, and 0.1% gentamycin (Sigma-Aldrich, St. Louis, MO). Cells were triturated at least 21 times to disperse cells, and cells were enumerated using a hemocytometer. Cells were seeded on collagen-coated 12-well plates at 0.1 × 106 cells/well for proliferation assays and at 0.4 × 106 cells/well for differentiation studies. Plates were incubated at 5% CO2 at 37◦ C. Twenty-four h after being plated, media were switched to McCoy 5A medium (Thermo Fisher Scientific, Gibco, Gaithersburg, MD) containing 10% chicken

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MUSCLE SATELLITE CELL BEHAVIORS

serum, 5% horse serum, 1% pen/strep, and 0.1% gentamycin.

BrdU Incorporation and Detection Forty-eight h after isolation, bromodeoxyuridine (BrdU) labeling reagent (Invitrogen, Carlsbad, CA) was added to each well at a 1:100 dilution. Cultures were allowed to incubate at 37◦ C for 1 h, after which media were discarded, and cell monolayers were washed once with ice-cold PBS, fixed in 1 mL of ice-cold 70% ethanol for 5 min at room temperature, and washed with PBS. After removal of PBS, plates were treated with 0.5 mL of 1.5 M hydrochloric acid and allowed to sit at room temperature for 30 minutes. Plates were washed twice with PBS and blocked in PBS with 5% goat serum (Thermo Fisher Scientific, Gibco, Gaithersburg, MD) for 1 hour. Plates were then incubated with an anti-BrdU antibody (clone G3G4, DSHB, Iowa City, IA) diluted 1:100 in PBS containing 5% goat serum. Plates were allowed to incubate overnight at 4◦ C. The following d, plates were washed 3 times with PBS, and a secondary antibody, Alexa Fluor 555 goat anti-mouse IgG (Life Technologies, Eugene, OR), diluted 1:1000 in PBS containing 5% goat serum, was applied. Cultures were incubated in the dark at room temperature for 2 hours. Plates were washed in PBS, and a fluorescent mounting medium was added to each well. 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) staining was used to identify nuclei. Images were collected using a Nikon ECLIPSE Ti-E fluorescent microscope (Nikon Instruments Inc., Melville, NY). Number of nuclei positive for BrdU was quantified as a percent of total number of nuclei, and the percentage was used as an indicator for cell proliferation rate.

Myogenic Differentiation Cells were plated at a density of 95% confluency to assess differentiation (106 cells/mL/well) in 12-well plates. To induce differentiation, the growth medium was switched to differentiation medium (high-glucose DMEM containing 3% horse serum and 1% pen/strep), and allowed to incubate at 37◦ C for 24 hours. Plates were washed once with ice-cold PBS and fixed with 1 mL of ice-cold 100% methanol at room temperature for 10 min. Plates were washed in PBS, blocked, and stained using a primary antibody against myosin (clone MF20, DSHB, Iowa City, IA) and a secondary antibody, Alexa Fluor 555 goat anti-mouse IgG. Plates were incubated at room temperature in the dark for 2 hours. Plates were washed with PBS, and fluorescent mounting medium was added to each well. DAPI staining was used to identify nuclei. Images were taken using a Nikon ECLIPSE Ti-E fluorescent microscope (Nikon Instruments Inc., Melville, NY) and analyzed using ImageJ software. Myotube diameter was determined by measuring the diameter at 3 separate points per myotube

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and averaged. Fusion index was calculated as a percentage of nuclei within the myotubes to total nuclei. Myonuclear domain was measured as myotube area in μm2 per nucleus.

Muscle Histochemistry Cyrosectioned muscle samples on silane-coated slides were allowed to dry at room temperature for 30 min prior to staining. Sections were stained with hematoxylin for 6 min then rinsed with running deionized water for 5 minutes. Slides were submerged in eosin for 2 s and then rinsed with running deionized water. Slides were rinsed in 50 and 70% ethanol 10 times, then 95% ethanol for 30 s, and then 100% ethanol for 60 seconds. Slides were finally rinsed in xylene 7 times and dried with a Kimwipe. Slides were cover and mounted in Permount mounting medium (Thermo Fisher Scientific, Waltham, MA). Random images were taken using a Nikon ECLIPSE 80i light microscope (Nikon Instruments Inc., Melville, NY). ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze images.

Muscle Immunohistochemistry Frozen muscle cyrosections on silane-coated slides were dried at room temperature for 30 min prior to staining. Slides were washed twice in PBS, then fixed in 4% paraformaldehyde for 10 min at room temperature. Sections were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS at room temperature for 15 min then incubated in blocking solution (5% goat serum in PBS) for 30 min at room temperature. Blocking solution was removed with a pipette and the primary antibodies Pax7 (1:50 dilution, DSHB, Iowa City, IA), Myf5 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), and/or eMyHC (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in blocking solution and added to slides and left overnight at 4◦ C. The following d, slides were washed 3 times with PBS, and secondary antibodies (Alexa Fluor 555 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG), including DAPI, were diluted 1:1000 and 1:500, respectively, in blocking solution and applied for 2 h in the dark at room temperature. Sections were washed 3 times in PBS, then mounted with fluorescent mounting medium. Images were taken using a Nikon ECLIPSE Ti-E fluorescent microscope (Nikon Instruments Inc., Melville, NY).

Muscle Injury The PM muscles of 91 d HWS and LWS males were damaged by intramuscular injection of 2% barium chloride (BaCl2 ) dissolved in saline solution. The HWS received 1 mL, and the LWS received an equivalent amount normalized for BW. Birds received BaCl2

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Figure 1. Muscle fibers from HWS chickens are larger than those from LWS chickens. Biceps femoris (BF) and pectoralis major (PM) muscles were isolated from 56-day-old HWS and LWS chickens. (A) Fluorescent images showing the nuclei (DAPI-stained, blue) and fiber (green). Scale bar, 50 μ m. (B) Muscle fiber size of BF and PM muscles. (C) Histograms showing the distribution of muscle fibers vs. fiber size in BF and PM muscles. N = 6 from each genotype. Data represent means ± SEM. ∗∗∗ P < 0.001 compared to HWS.

injections on one side of the PM muscle and contralateral saline solution injections on the opposite muscle 7, 3, and 1 d prior to harvest. To track the injection sites, BaCl2 and saline solutions were mixed with 1% India ink. Samples were collected and sections were made as outlined previously.

Statistical Analysis Data are presented as means ± standard error of the mean (S.E.M.), with significance set as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Data analysis was conducted using Excel Student t test.

RESULTS To evaluate the effects of long-term selection for BW on muscle fiber size, 6 males were selected from each line at 56 d of age, and muscle samples from the BF (slow-twitch, red, and oxidative) and the PM (fasttwitch, white, and glycolytic) were collected, sectioned, and stained. The BF and PM muscles from HWS had larger myofibers compared to LWS (Figure 1A). Quantitatively, the cross-sectional areas of the myofibers

were considerably larger in muscles of HWS than LWS (Figure 1B). Consistently, histograms of muscle crosssectional areas corroborated findings that muscle of HWS had larger fibers in BF and PM muscles, respectively (Figure 1C). Together, these findings show that muscles of chickens selected for larger body size possess larger myofibers. To investigate the potential differences in SC abundance and quiescence status between muscle of HWS and LWS, we stained BF and PM muscle cryosections from 56 d males with Pax7 and Myf5 antibodies. Pax7+; Myf5- cells represent quiescent SC, while Pax7+; Myf5+ cells indicate activated SC and Pax7-; Myf5+ cells are committed myogenic SC (Halevy et al., 2004; Bentzinger et al., 2012; Gurevich et al., 2016). Muscle of HWS had significantly more SC per myofiber in both the BF and PM muscles than LWS (Figure 2B). Muscle from the BF of HWS contained a higher percentage of activated (Pax7+; Myf5+) and committed (Pax7-; Myf5+) SC subpopulations, whereas the quiescent (Pax7+; Myf5-) SC subpopulation was proportionally lower than that of LWS (Figure 2C). In contrast, there was no difference in quiescent SC subpopulation between the 2 lines, although

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Figure 2. SC content and subpopulation in BF and PM muscles. (A) Representative images showing the staining of Pax7 (red), Myf5 (green), and nuclei (DAPI, blue). Red arrow, Pax7+ only; green arrow, Myf5+ only; yellow arrow, Pax7+ Myf5+ . Scale bar, 50 μ m. (B) SC number per 100 muscle fibers. SC number was calculated as the total of Pax7+ + Myf5+ + Pax7+ Myf5+ cells. (C) SC subpopulation in HWS and LWS muscles. Pax7+ , Myf5+ , and Pax7+ Myf5+ cell subpopulations were calculated as percent of total. (D) Nuclear number per muscle fiber was counted and compared between HWS and LWS chickens. N = 6 from each genotype. Data represent means ± SEM. ∗ P < 0.05, ∗∗∗ P < 0.001 compared to HWS.

HWS SC still contain a higher percentage of activated SC in the PM (Figure 2C). We enumerated the number of nuclei per muscle fiber and found that myofibers of HWS birds contain more nuclei per muscle fiber than those prepared from LWS muscle, regardless of muscle (Figure 2D). Because SC are the major contributor of myonuclei accretion post hatch, these data suggest myofibers of HWS result from greater SC accretion, perhaps through SC that are more likely to be activated. To test further the functions of SC derived from HWS and LWS muscles, we isolated and cultured SC in vitro. This was accomplished using 5-day-old male chicks to avoid growth-related pressure on SC proliferative properties most likely expended through muscle growth as the chicken ages. SC were pulsed 1 h with BrdU, a thymidine analog, in growth medium to evaluate proliferation. A higher number of SC derived from HWS incorporated BrdU than those collected from LWS (Figure 3B). The percentage of proliferating cells was quantified by dividing the number of BrdU+ nuclei by the total number of nuclei and served as an indicator of SC proliferative capacity. The SC proliferative capacity was greater in HWS compared to LWS (Figure 3C and D).

To test the difference in myogenic differentiation between SC from HWS and LWS, cells were isolated again from 5-day-old male chicks. An equal number of SC were plated and induced to differentiate for one day. Afterwards, differentiation cells were stained with a myosin antibody to identify myotubes and assess differentiation. SC derived from HWS muscles had more robust differentiation capacity, as reflected by a greater staining of the differentiation marker, myosin (Figure 4A). Quantitatively, HWS SC produced larger myotubes with significantly larger diameters than LWS SC (Figure 4B). Along with larger myotubes, HWS SC exhibited a higher fusion index compared to LWS SC (Figure 4C). Myonuclear domain is defined by the ratio of the muscle fiber cross-sectional area to the nuclear number within a given area and is often used to measure the functionality and efficiency of each nucleus (White et al., 2010). We observed that HWS myotubes had a significantly larger nuclear domain than LWS myotubes (Figure 4D). Together, these results show that SC from HWS muscle have enhanced capacities to differentiate. To confirm the in vitro differences in properties of SC derived from HWS and LWS muscles in vivo, barium chloride injection was used to create a muscle

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Figure 3. SC from HWS chicken exhibit higher proliferative capacity. (A) Identification and quantification of SC. SC were stained with Myf5 (green) and counterstained with DAPI (blue). Scale bars, 50 μ m. (B) SC were isolated from 5-day-old chickens and cultured in growth medium for 3 days. 10 μ M of BrdU was added to the growth medium for 1 h; cells were fixed and stained with BrdU antibody. Nuclei were stained with DAPI (blue). Scale bars, 50 μ m. (C-D) Quantification of BrdU+ cells expressed as percent of total number of nuclei. Data represent mean ± SEM from 3 independent experiments. ∗∗∗ P < 0.001 compared to the HWS.

damage/regeneration model. Damaged muscles from 91 d males were collected 3 d after injury and stained with Myf5 to identify proliferating SC, and embryonic myosin heavy chain (eMyHC) to identify differentiating SC (Figure 5A). Damaged HWS muscle had significantly more proliferating SC than that in LWS in the damaged area as assessed by immune-reactivity SC markers (Figure 5B). Similarly, more eMyHC+ muscle cell staining was detected in HWS than LWS muscle suggesting greater myogenic differentiation and presumably nascent myofiber fiber development, though

the latter is conjecture (Figure 5C). Together, these in vivo data support in vitro data by showing HWS SC have greater abilities to activate, proliferate, and differentiate than those SC of LWS muscle. To study the contribution of SC to adult regenerative myogenesis, we harvested the muscles 7 d post injury and found that HWS exhibited a more robust regeneration than LWS (Figure 6A). Cross-sectional areas of nascent myofibers were larger in the HWS than LWS muscle (Figure 6B). Differences were illustrated in histograms showing that the distribution of HWS myofiber

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Figure 4. SC derived from HWS exhibited enhanced differentiative capacity. SC were isolated from HWS and LWS in 5-day-old chickens. Equal number of SC were plated and induced to differentiate for one day. Myotubes were stained with DAPI for nuclei and MF20 for myosin. (A) Representative images of SC differentiation from HWS and LWS chickens. (B-D) Diameter (B), fusion index (C), and myonuclear domain (D) of myotubes. Scale bar, 200 μ m. Data represent mean ± SEM from 3 independent experiments. ∗∗∗ P < 0.001 compared to HWS.

size shifted to the right (larger size) (Figure 6C). Taken together, these data show a greater capacity to regenerate muscle in HWS than LWS and support the notion that chickens selected long term for BW have muscle tissue with enhanced SC capacity to support increased muscle growth and regeneration.

DISCUSSION Long-term selection for growth undoubtedly results in dramatic alterations in whole body metabolism and myriad changes in tissue growth dynamics (Velleman et al., 2000; Rehfeldt et al., 2002; Yin et al., 2014). For example, HWS and LWS chickens differ considerably in muscle growth rate, feed consumption, feed utilization, and various muscle gene expression patterns (Dunnington and Siegel, 1996; Yin et al., 2014). HWS chickens used in this study accrete more muscle protein than LWS as reflected in the increased muscle fiber cross-sectional area. This trend also is observed in culture in which SC from HWS muscle not only have enhanced capacities to differentiate but are also more robust in terms of supporting protein synthesis and muscle cell hypertrophy, as evidenced by enhanced nuclear domain in HWS myotubes. The underlying mechanisms

responsible for developing such a disparity in muscle mass due to selection is intriguing. SC are a major contributor to post-hatch muscle growth as well as tissue maintenance and repair in a wide range of species (Muir et al., 1965; Reger and Craig, 1968; Kryvi, 1975; Seger et al., 2011). Because SC are the major driving force for continued and necessary DNA accretion in growing muscle, it is not surprising to find that HWS and LWS SC differ in their intrinsic properties to activate, divide, and ultimately add nuclei to existing muscle fibers. Although the ultimate biology responsible for this divergent phenotype is not known, it is likely that selection for BW is a direct result of more cells produced in ovo. From a developmental prospective, muscle progenitor cells migrate from the somite to populate various organizational centers, which ultimately develop muscle (Brack and Rando, 2012). Satellite cells represent a subset of these cells. If greater myogenic precursors leave the somite, it is likely that a greater number of SC become trapped between the basal lamina and cell membrane of developing fibers. To this end, more SC would be available to support greater tissue growth. Alternatively, growth rate is also partly a function of the time needed to replicate cells. Clearly, HWS SC

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Figure 5. SC from HWS chickens exhibit enhanced myogenesis in vivo. PM muscle was damaged by BaCl2 injection. (A) Three d after injury, muscles were harvested and stained for Myf5 (red), embryonic MyHC (green), and nuclei (DAPI, blue). Red arrow, Myf5+; green arrow, eMyHC+. Scale bar, 25 μ m. (B) Quantification of Myf5+ cells per mm2 . (C) Quantification of embryonic myosin heavy chain positive (eMyHC +) cells per mm2 . N = 6 in each genotype. Data represent mean ± SEM. ∗∗∗ P < 0.001 compared to HWS.

retain the ability to proliferate more rapidly than those originating from LWS. Moreover, the time necessary to proceed through the cell cycle may be shorter, thereby decreasing the time necessary to grow the same or more tissue mass. Because HWS chickens contain more SC that consist of a larger proportion of activated cells than LWS, HWS muscle possesses a larger reservoir of activated cells ready to contribute their nuclei to existing myofibers. This hypothesis is strongly supported by the observations that HWS muscle recovered faster at d 7 post injury than LWS muscle when presented with a chemical insult. Given SC are the major contributor of regenerative myogenesis, intrinsic differences between HWS and LWS SC are likely important drivers of the phenotypical differences in growth observed between these 2 distinct chicken lines. However, it does not exclude the possibility that factors such as extracellular matrix, hormones, and other growth factors may contribute to the distinctions of in vivo regenerative myogenesis observed in HWS and LWS chickens, yet inherent differences in SC clearly exist ex vivo. Another observation is the difference of SC abundance and homeostatic status between red and white muscle. Red muscles are predominantly oxidative in

terms of producing energy and thus require extensive oxygen availability to support mitochondrial betaoxidation of fatty acids to supply energy for contraction. Alternatively, type II, glycolytic, and fast-twitch muscles experience a much different microenvironment characterized by a smaller capillary network and fewer fiber mitochondria than their type I fatigue-resistant counterparts (Pette and Staron, 2000; Bassel-Duby and Olson, 2006; Shi et al., 2007; Shi et al., 2008; Shi et al., 2009). As a result of these different physiologies, it is reasonable to postulate that red and white muscles provide unique niches by which resident SC are patterned for particular behaviors and fates. Several lines of evidence support this notion. First, in vitro studies show that SC derived from red and white muscles have divergent gene expressions (Barjot et al., 1993; Martelly et al., 2000; Brzoska et al., 2009), fate decisions (Yada et al., 2006), activation status (Fig. 2B and C), and differentiation profiles (Feldman and Stockdale, 1991; Barjot et al., 1995; Rosenblatt et al., 1996). Second, because SC isolated from red and white muscles can be forced to replicate in vitro several times, ultimately they differentiate into myotubes that resemble the nature of their host myofibers (Dusterhoft and Pette, 1993;

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99 99 99 99 99 0-5 0-8 0-11 00-14 00-17 30 60 90 15 12 Cross sectional area (µm2)

Figure 6. HWS chicken muscle exhibited improved regeneration. PM muscles from 91-day-old chickens were injected with 2% barium chloride (BaCl2) to induce injury. Muscle samples were harvested 7 d post injury. (A) H&E staining. Scale bars, 100 μ m. (B-C) Muscle fiber cross sectional area (B) and fiber size distribution histogram (C) of 7 d newly formed fibers. ∗∗∗ P < 0.001 compared to HWS.

Barjot et al., 1995; Rosenblatt et al., 1996). Third, it has been demonstrated that red and white muscles contain differing numbers of SC that self-renew differently (Christov et al., 2007; Manzano et al., 2011). Fourth, SC behaviors change with altered nutritional signals. Diabetes mellitus, e.g., impairs regenerative myogenesis by compromising SC activation (D’Souza et al., 2013), while calorie restriction enhances SC proliferation and influences the efficiency of SC engraftment (Cerletti et al., 2012). Although both BF and PM muscles’ myofibers are larger in size and contain more SC in HWS than LWS myofibers, the former harbor more SC than PM. These results are consistent with those

reporting that red muscles contain more SC than white muscles (Schmalbruch and Hellhammer, 1977; Gibson and Schultz, 1982). Moreover, when SC pools are segregated into quiescent, activated, and myogenic subpopulations, we find differences between red and white muscles. Specifically, BF muscle contains less quiescent but more activated and committed SC subpopulations than PM muscle in HWS chickens, whereas disparities in SC subpopulations from muscles of LWS chickens were less noticeable. Obviously, niche-based factors from red vs. white muscles, and HWS and LWS lines are important in governing SC behaviors in their respective niches,

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although the molecular mechanism behind it remains unknown. Although we show that SC from HWS and LWS chickens differ in a number of characteristics, significant questions remain to be addressed. For example, if SC are restricted to a set number of divisions (Sacco et al., 2010; Shi et al., 2013) will SC pools be exhausted sooner in HWS than LWS muscle fibers? Further, what are the genetic and/or epigenetic modifications that determine the different behaviors of the isolated SC in vitro? Can we boost muscle growth if we transplant the HWS SC into LWS muscles? Answers to these questions will undoubtedly aid our understanding of how niches shape SC behaviors, which, in turn, dictate the speed and status of muscle growth and repair.

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