Published December 2, 2014

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1 Published December 2, 2014 MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Manipulating mesenchymal progenitor cell differentiation to optimize performance and carcass value of beef cattle 1,2 M. Du,* 3 Y. Huang, A. K. Das,* Q. Yang,* M. S. Duarte,* M. V. Dodson,* and M.-J. Zhu *Department of Animal Sciences, Washington State University, Pullman 99164; Department of Animal Science, University of Wyoming, Laramie 82071; and School of Food Science, Washington State University, Pullman ABSTRACT: Beef cattle are raised for their lean tissue, and excessive fat accumulation accounts for large amounts of waste. On the other hand, intramuscular fat or marbling is essential for the palatability of beef. In addition, tender beef is demanded by consumers, and connective tissue contributes to the background toughness of beef. Recent studies show that myocytes, adipocytes, and fibroblasts are all derived from a common pool of progenitor cells during embryonic development. It appears that during early embryogenesis, multipotent mesenchymal stem cells first diverge into either myogenic or adipogenic-fibrogenic lineages; myogenic progenitor cells further develop into muscle fibers and satellite cells whereas adipogenic-fibrogenic lineage cells develop into the stromal-vascular fraction of skeletal muscle where reside adipocytes, fibroblasts, and resident fibro-adipogenic progenitor cells (the counterpart of satellite cells). Strengthening myogenesis (i.e., formation of muscle cells) enhances lean growth, promoting intramuscular adipogenesis (i.e., formation of fat cells) increases marbling, and reducing intramuscular fibrogenesis (i.e., formation of fibroblasts and synthesis of connective tissue) improves overall tenderness of beef. Because the abundance of progenitor cells declines as animals age, it is more effective to manipulate progenitor cell differentiation at an early developmental stage. Nutritional, environmental, and genetic factors shape progenitor cell differentiation; however, up to now, our knowledge regarding mechanisms governing progenitor cell differentiation remains rudimentary. In summary, altering mesenchymal progenitor cell differentiation through nutritional management of cows, or fetal programming, is a promising method to improve cattle performance and carcass value. Key words: adipogenesis, beef, fibrogenesis, marbling, myogenesis, progenitor cells 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas INTRODUCTION According to the past surveys of beef producers by National Cattlemen s Beef Association, marbling and tenderness were consistently identified as the top beef quality problems (McKenna et al., 2002; Garcia 1 Based on a presentation at the Meat Science and Muscle Biology Symposium titled In utero factors that influence postnatal muscle growth, carcass composition, and meat quality at the Joint Annual Meeting, July 15 19, 2012, Phoenix, AZ, with publication sponsored by the Journal of Animal Science and the American Society of Animal Science. 2 This work was supported by Agriculture and Food Research Initiative Competitive Grant no from the USDA National Institute of Food and Agriculture. 3 Corresponding author: min.du@wsu.edu Received July 20, Accepted September 7, et al., 2008). Marbling becomes a top quality problem because of the selection for increased lean growth, which results in overall reduction of fat accumulation, including intramuscular fat (i.e., marbling) that is critical for the palatability of meat (Du et al., 2010a). Beef tenderness is mainly determined by 2 factors: myofibrils and connective tissue. Myofibrillar effect on beef tenderness can be largely solved by proper aging of carcasses and stretching of muscle fibers. On the other hand, connective tissue is responsible for the background toughness. Because connective tissue and its cross-linking resist protease hydrolysis, postmortem aging is not effective in tenderizing beef with greater connective tissue content (Kuber et al., 2004; Lepetit, 2008), which leaves the reduction of connective tissue content and its cross-linking as the choice for

2 1420 Du et al. alleviating background toughness of beef. Given that myogenic, adipogenic, and fibrogenic cells are derived from a common pool of mesenchymal progenitor cells, manipulation of progenitor cell differentiation provides a unique opportunity to enhance lean growth, reduce connective tissue accumulation, and alter fat deposition, with the objective of improving the efficiency and quality of meat production. Here, we first discuss the differentiation of mesenchymal progenitor cells into myogenic, adipogenic, and fibrogenic cells as well as known mechanisms governing their differentiation, focusing on adipogenesis and fibrogenesis. Then we introduce practical approaches to manipulate mesenchymal progenitor cell differentiation, including the concept of fetal programming and its possible application to animal production. SKELETAL MUSCLE, ADIPOSE, AND CONNECTIVE TISSUE DEVELOPMENT Overview of Skeletal Muscle, Intramuscular Adipose, and Connective Tissue Development Skeletal muscle development is roughly divided into 3 stages: embryonic, fetal, and adult stages (Du et al., 2010a). Embryonic and fetal skeletal muscle development is collectively referred to as prenatal muscle development, which is critical because it has dramatic impact on postnatal growth (Dauncey and Harrison, 1996). During the prenatal stage, skeletal muscle development mainly involves the formation of muscle fibers (i.e., myogenesis) but also the formation of intramuscular adipocytes (i.e., adipogenesis) and fibroblasts (i.e., fibrogenesis). In livestock, muscle fibers are formed during the prenatal stage and there is no further net increase after birth. Intramuscular adipogenesis during early developmental stages generates sites for fat deposition in offspring muscle, which forms marbling fat in resulting beef; adipogenesis in other depots is undesirable. Intramuscular fibrogenesis creates connective tissue distributed inside muscle. Recent studies show that intramuscular adipocytes and fibroblasts are developed from common progenitor cells (Joe et al., 2010; Uezumi et al., 2010, 2011). As a result, intramuscular adipogenesis and fibrogenesis may be considered as a competitive process, providing that the total density and proliferation of progenitor cells are unaltered; enhancing adipogenic differentiation and reducing fibrogenic differentiation from progenitor cells will increase both the marbling and tenderness of meat. Skeletal Muscle Development During the prenatal stage, myogenesis is separated into primary myogenesis during early gestation and secondary myogenesis during mid to late gestation in pigs (i.e., up to around 90 d, with term being 114 d; Wigmore and Stickland, 1983) and mid gestation in cattle (i.e., up to around 180 d, with term being 284 d; Bonnet et al., 2010). The primary myogenesis occurs during the embryonic stage, which forms the templates for secondary myogenesis during the fetal stage (Swatland, 1973). Secondary myogenesis and further increase in muscle fibers at the late fetal stage form the majority of muscle fibers. Therefore, the prenatal stage, especially mid gestation, is critical for skeletal muscle development (Greenwood et al., 2000; Du et al., 2010b). Because muscle fibers result from the fusion of myogenic cells, greater abundance of myogenic cells due to active proliferation results in more muscle fiber formation during the fetal stage (Zhu et al., 2004). However, the proliferation of myogenic precursor cells is highly sensitive to nutrients and endocrine regulation; thus, maternal nutrition and physiological condition affect the proliferation and abundance of myogenic cells and the subsequent formation of muscle fibers (Zhu et al., 2004, 2008; Tong et al., 2009; Yan et al., 2010). One important regulator of myogenic cell proliferation is myostatin, which inhibits proliferation to reduce muscle fiber numbers, and its mutation consistently results in double muscling in cattle (McPherron and Lee, 1997). Postnatal muscle growth is mainly due to the increase in muscle fiber size (Brameld et al., 2000), which relies on muscle satellite cells. Muscle satellite cells, originating from the embryonic myotome, lie between the sarcolemma of myofibers and surrounding basal lamina in adult skeletal muscle (Reznik, 1969). The majority of nuclei in adult muscle fibers are derived from muscle satellite cells (Allen et al., 1979), showing the critical role of these cells for postnatal muscle growth. However, if there is insufficient muscle fiber formation during the fetal stage, postnatal muscle growth is severely constrained due to the lack of muscle fibers because the size of muscle fibers cannot exceed certain limits that allow efficient exchange of nutrients and metabolites. Indeed, runt piglets, which do not obtain sufficient nutrients for muscle fiber formation, have less muscle mass permanently (Aberle, 1984; Handel and Stickland, 1987). Satellite cells are committed myogenic cells. Besides satellite cells, multipotent progenitor cells, such as pericytes, are able to differentiate into myogenic cells, thereby contributing to postnatal muscle growth (Dellavalle et al., 2011).

3 Progenitor cells in beef production 1421 Molecular Regulation of Myogenesis Regardless of prenatal or postnatal skeletal muscle development, myogenesis is under the control of a number of regulatory proteins, including wingless and integration 1 (Wnt), paired box gene (Pax) 3 and Pax 7, and myogenic regulatory factors (MRF; Maroto et al., 1997; Hyatt et al., 2008). The Wnt signaling plays a crucial role in activating myogenic differentiation (Cossu and Borello, 1999). The expression of Pax 3 and Pax 7 in progenitor cells induces the expression of MRF (Bailey et al., 2001), including MRF-4, myogenic differentiation 1 (MyoD), myogenic factor 5 (Myf5), and myogenin (Bergstrom and Tapscott, 2001; Stewart and Rittweger, 2006). Two of these factors, MyoD and Myf5, function compensatively to induce the differentiation of precursor cells into myoblasts (Roth et al., 2003). Myogenin is important for the fusion of myoblasts into myotubes (Barnoy and Kosower, 2007) whereas MRF-4 appears to be required to maintain the myogenic identity of muscle cells (Kassar-Duchossoy et al., 2004). ADIPOGENESIS AND ADIPOSE TISSUE DEVELOPMENT Overview of Adipose Tissue and Adipogenesis Adipose tissue is not only an energy storage site (MacDougald and Mandrup, 2002) but also an endocrine organ that secretes adipokines (Lundgren et al., 1996; Alessi et al., 1997; Camp et al., 2002; Feve, 2005; Ailhaud, 2006; Avram et al., 2007), through which it regulates whole body energy balance (Kahn and Flier, 2000; Trujillo and Scherer, 2006). There are 4 major adipose tissue depots in livestock: the visceral, subcutaneous, intermuscular, and intramuscular depots. Whereas the accumulation of adipose tissue intramuscularly is highly desirable, accumulation of adipose tissue elsewhere is a liability to livestock production, dramatically increasing production costs. It is a common observation that certain animals have much greater marbling than their counterpart animals; however, to date, mechanisms leading to the preferential accumulation of intramuscular fat remain poorly defined. Adipocytes, especially intramuscular adipocytes, share immediately common progenitor cells with myogenic cells during fetal muscle development. As a result, exploring mechanisms governing the early commitment of progenitor cells to either myogenic or adipogenic-fibrogenic lineages will help us to understand the preferential formation of intramuscular adipocytes and the resulting marbling. Consistent with this notion, a growing body of evidence shows that intramuscular adipocytes behave differently compared with subcutaneous and visceral adipocytes (Gardan et al., 2006; Zhou et al., 2007; Hausman et al., 2008; Rajesh et al., 2010); thus, the unique developmental origin and properties of intramuscular adipocytes provide targets to specifically enhance marbling without elevating overall obesity of animals. Sequential Adipose Tissue Development. The formation of discernible adipocytes and adipose tissue begins before mid gestation in beef cattle (Bonnet et al., 2010), with the first detection of adipocytes in visceral fat depots followed by subcutaneous, intermuscular, and intramuscular fat depots. In perinatal fat, adipocytes are detectable as early as 80 d of gestation whereas adipocytes in the intermuscular fat are detectable at 180 d of gestation (Taga et al., 2011). Most adipocytes are formed during the fetal and early postnatal stages, and adipocyte hyperplasia largely ceases in perirenal fat after birth (Bonnet et al., 2010; Du et al., 2010b). It is suggested that the total number of adipocytes is set when reaching adolescence (Goessling et al., 2009). As for livestock, they are generally slaughtered at a young age and, therefore, adipocyte hyperplasia is ongoing lifelong but dwindles as animals become older; adipose tissue development in growing cattle is mainly due to hypertrophy (Robelin, 1981; Cianzio et al., 1985). The dwindling presence of progenitor cells as animals age is the major reason for the declining ability to generate new adipocytes (Du et al., 2010c). Therefore, nutritional and physiological conditions during the fetal, postnatal, and early postweaning stages affect adipogenesis and the total number of adipocytes in animals (Fig. 1). The sequential formation of adipocytes in these 4 different depots provides an opportunity to preferentially enhance intramuscular adipogenesis over other fat depots. In Fig. 1, we outline the time course for the formation of these 4 fat depots. The formation of adipocytes in the visceral depot occurs during the mid fetal stage to early postnatal stage (Robelin, 1981); the formation of subcutaneous adipocytes occurs slightly later, between the mid to late fetal stage and the early weaning stage (Hood and Allen, 1973). The formation of intramuscular adipocytes, however, has not been systemically studied and is estimated to mainly occur during the late fetal neonatal stage to about 250 d of age in beef cattle (Fig. 1). As a result, there is a unique time window to specifically enhance marbling without an overall increase in fatness. Supplementation of nutrients or other bioactive compounds to enhance adipogenesis during the early weaning stage to about 250 d of age is expected to specifically enhance intramuscular adipogenesis, which provide sites for lipid accumulation during the fattening stage, resulting in adipocyte hypertrophy and high marbling (Fig. 1). This notion is supported by several previous studies with beef cattle that identify early weaning to around 250 d of age as the

4 1422 Du et al. marbling window (Wertz et al., 2001, 2002; Pyatt et al., 2005a,b; Corah and McCully, 2007). There are studies indicating that feeding early-weaned calves a grainbased (especially corn-based) diet increases marbling (Wertz et al., 2001, 2002; Pyatt et al., 2005a,b). Mechanisms Governing Adipogenesis Adipogenesis is a term that describes the de novo generation of adipocytes, which is briefly separated into 2 stages: determination and differentiation (MacDougald and Mandrup, 2002). Most studies on adipogenesis focus on differentiation, in other words, the conversion of preadipocytes into mature adipocytes. During this stage, PPARγ and C/EBP have critical regulatory roles (Avram et al., 2007). Another transcription factor, C/EBPβ/δ, which is expressed at the very early stage of adipogenesis, triggers the expression of PPARγ (Fajas et al., 2001), an essential and indispensable transcription factor for the late stage of adipogenesis; PPARγ and C/EBPα reinforce each other to induce genes specific to adipocytes (Spiegelman and Flier, 1996; Rosen and MacDougald, 2006). As a result, cells accumulate lipid droplets and become mature adipocytes (Brun and Spiegelman, 1997). Because most studies on adipogenesis were conducted in 3T3-L1 cells, which are preadipocytes, our understanding of the formation of preadipocytes, or the initial determination stage of adipogenesis, is quite limited. Recently, zinc-finger protein (Zfp) 423 was identified as a transcriptional factor responsible for the adipogenic commitment of progenitor cells (Gupta et al., 2010). The expression of Zfp423 commits progenitor cells to preadipocytes, which further induces PPARγ expression and terminal adipogenic differentiation of cultured NIH 3T3 cells and in subcutaneous fat (Gupta et al., 2010, 2012). CONNECTIVE TISSUE DEVELOPMENT Overview of Connective Tissue Development and Meat Tenderness Fibrogenesis usually refers to the generation of fibroblasts and the formation of fibrous connective tissue. Fibrogenesis is very active during the fetal stage, during which it generates connective tissue forming primordial perimysium and epimysium in fetal skeletal muscle during the late gestation (Du et al., 2010b). Fibrosis is defined by the enhanced deposition of extracellular matrix proteins, mainly collagens, in the basement membrane and interstitial tissue, and it is usually a pathological feature and marks the recovery stage of many diseases (Liu and Pravia, 2010). Connective tissue, mainly collagen, is responsible for the background toughness of meat. For this reason, tender beef is limited to muscles with low collagen content, such as LM, whereas beef derived from limb muscles has high collagen content and is tougher (McCormick, 1999). In addition to collagen content, collagen cross-linking has even greater impact on toughness of beef (McCormick, 1994). Lysyl oxidase is the critical enzyme regulating cross-linking of collagen fibrils (Borg et al., 1985; Huang et al., 2012). Available studies show that collagen content and cross-linking are positively correlated to Figure 1. Concept of marbling window based on the sequential formation of adipocytes in 4 major fat depots. The timelines for adipogenesis in different fat depots are approximate. See online version for figure in color.

5 Progenitor cells in beef production 1423 each other whereas collagen turnover is negatively correlated with cross-linking (Archile-Contreras et al., 2010). Collagen turnover is accelerated by compensatory growth and extracellular remodeling and is a process that increases tenderness (Hill, 1967; Archile-Contreras et al., 2011). Other components of connective tissue, such as decorin, which is a key component of basal lamina and extracellular matrix, have critical roles in mediating the biological effects of growth factors, progenitor cell differentiation, and overall muscle development (Velleman, 1999; Li et al., 2008). To dramatically reduce background toughness overall, preventing excessive collagen accumulation is the key. Mechanisms Regulating Fibrogenesis Although a number of cytokines and growth factors involved in the regulation of fibrogenesis, transforming growth factor (TGF)-β is the most important profibrogenic cytokine (Liu and Pravia, 2010). Three isoforms of TGF-β have been identified, which are TGF-β1, TGF-β2, and TGF-β3. The TGF-β1 isoform is primarily expressed in endothelial cells, fibroblasts, hematopoietic cells, and smooth muscle cells, TGF-β2 mainly exists in epithelial cells and neurons, and TGF-β3 is specifically expressed in mesenchymal cells (Ghosh et al., 2005). All TGF-β isoforms activate downstream Sma and Mad related proteins (SMAD) signaling (Attisano and Wrana, 1996; Letterio and Roberts, 1998). The SMAD family contains 5 receptor-regulated SMAD (R-SMAD 1, 2, 3, 5, and 8), a common SMAD (Co-SMAD4), and 2 inhibitor SMAD (I-SMAD 6 and 7; Moustakas et al., 2001). The ligand, TGF-β, first binds to TGF-β receptor (TβR) II, which then recruits and activates TβRI. Then SMAD2 and SMAD3 are phosphorylated and subsequently bind to SMAD4 (Suwanabol et al., 2011), and the resulting SMAD complex is translocated into the nucleus where it binds to SMAD-specific binding elements of target genes, thereby activating the expression of fibrogenic genes including procollagen and enzymes catalyzing collagen cross-linking (Massague and Chen, 2000). Collagen is the main component of connective tissue and the structural unit of extracellular matrix (Vanderrest and Garrone, 1991). In muscle, Type I and Type III are the most abundant collagen in extracellular matrix (Light et al., 1985). Collagens have a very low turnover rate, and they are degraded by the action of matrix metalloproteinases (MMP). The activity of MMP is inhibited by tissue inhibitor of metalloproteinase (Visse and Nagase, 2003). Lysyl oxidase is the key enzyme catalyzing collagen cross-linking. In our studies in sheep and cattle, the expression of collagens, lysyl oxidase, and MMP are correlated with each other, showing that all of these components are needed to synthesize extracellular connective tissue (Huang et al., 2012). ADIPOCYTES AND FIBROGENIC CELLS SHARE COMMON PROGENITOR CELLS It has been well established that stromal vascular cells are major sources of adipogenic cells in skeletal muscle (Hausman et al., 2002; Hausman and Poulos, 2004). Very recently, it was conclusively established in rodents that intramuscular adipocytes and fibroblasts share immediate common ancestor cells, termed mesenchymal progenitor cells (Uezumi et al., 2011). These cells are mainly located in the stromal-vascular fraction of skeletal muscle and are distinct from satellite cells (Joe et al., 2010; Uezumi et al., 2010). Based on these new discoveries, we propose that, during skeletal muscle development, mesenchymal stem cells first diverge to either myogenic progenitor cells or adipogenicfibrogenic progenitor cells. Fetal myogenic progenitors further develop into muscle fibers and satellite cells whereas fetal adipogenic-fibrogenic progenitors develop into stromal-vascular fraction of mature skeletal muscle in which reside adipocytes, fibroblasts, and resident progenitor cells (Fig. 2). Because adipogenic and fibrogenic cells share immediate common progenitor cells, this provides an opportunity for us to manipulate the differentiation of progenitor cells to favor adipogenesis. Enhancing adipogenesis in developing muscle increases intramuscular fat (i.e., marbling), thereby improving beef flavor. On the other hand, less fibrogenic differentiation reduces intramuscular connective tissue, thereby decreasing background toughness of beef. In combination, the eating quality of beef can be improved. However, the mechanisms determining adipogenic or fibrogenic differentiation remain to be defined. Because Zfp423 has been demonstrated to be a transcriptional factor responsible for the adipogenic commitment of progenitor cells in subcutaneous fat and NIH 3T3 cells (Gupta et al., 2010), we hypothesized that Zfp423 is also important for intramuscular adipogenesis. To evaluate this, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with the telomerase reverse transcriptase pci neo-hest2, and individual clones were selected by G418. A total of 288 clones (3 96 well plates) were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. The expression of Zfp423 was much greater (307.4 ± 61.9% of control values; P < 0.05) in high adipogenic cells compared with low adipogenic cells. After adipogenic differentiation, the expression of

6 1424 Du et al. PPARγ and C/EBPα were much greater (239.4 ± 84.1% and ± 138.4% of control values, respectively; P < 0.05) in high adipogenic cells (Y. Huang, A. K. Das, Q. Yang, M.-J. Zhu and M. Du, unpublished data). Overexpression of Zfp423 in stromal-vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation. On the other hand, knockdown of Zfp423 attenuated adipogenic capacity of high adipogenic cells. These data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular adipogenesis and marbling in beef cattle and reducing intramuscular collagen accumulation and toughness of beef. Besides Zfp423, other regulators committing progenitor cells to adipogenic lineage likely exist and await further investigation. MANIPULATING PROGENITOR CELL DIFFERENTIATION THROUGH FETAL PROGRAMMING Fetal programming, also called developmental programming or the Barker hypothesis, refers to the fetal origins of adult diseases. Based on epidemiological data, this hypothesis indicates that low birth weight, due to maternal malnutrition among other factors, is associated with lasting effects on adult health (Barker, 2002) and that the changes in maternal uterine environment as a result of nutritional stress at certain stages of fetal growth and development might permanently affect tissue structure and function in offspring (Drake and Walker, 2004). Compared with brain and heart tissues, skeletal muscle has a lesser priority for nutrient repartitioning, which makes the development of skeletal muscle especially vulnerable to nutritional change (Zhu et al., 2006). Studies with a sheep model indicated that both maternal under- and overnutrition affected skeletal muscle development and intramuscular adipogenesis (Stannard and Johnson, 2004; Quigley et al., 2005; Tong et al., 2008, 2009; Zhu et al., 2008; Yan et al., 2010) and have long-lasting irreversibly negative physiological consequences for offspring. This phenomenon has been proven in in-utero undernourished lambs (Zhu et al., 2006), pigs (Dwyer et al., 1994), and guinea pigs (Ward and Stickland, 1991). Limited studies indicate that maternal nutrition affects fibrogenesis in skeletal muscle. For example, maternal nutrient restriction in swine increases collagen content in skeletal muscle of offspring (Karunaratne et al., 2005). In our studies, maternal overnutrition increased intramuscular fibrogenesis in skeletal muscle of sheep (Huang et al., 2010; Yan et al., 2011). Because fetal muscle development of ruminant animals mainly occurs during early to mid gestation, nutrient deficiency during this stage reduces muscle fiber numbers. At the late gestation stage, maternal nutrient restriction does not have major impacts on the number of muscle fibers in cattle and sheep, because skeletal muscle has matured at 105 d of gestation in sheep and 210 d in cattle (Du et al., 2010a). However, maternal nutrient restriction at this stage does reduce muscle fiber size (Greenwood et al., 1999) as well as postnatal muscle growth by reducing the population density of satellite cells (Woo et al., 2011). Because the late Figure 2. Commitment of mesenchymal progenitor cells into myogenic and fibro-adipogenic cell lineages during fetal muscle development. See online version for figure in color.

7 Progenitor cells in beef production 1425 gestation and early postnatal stages are also important for adipogenesis, maternal nutrition during pregnancy and nursing affects adipogenesis and, thus, the overall adiposity and marbling of offspring cattle, which has been described in our previous review (Du et al., 2011). SUMMARY AND CONCLUSIONS Myogenic, adipogenic, and fibrogenic cells are derived from common mesenchymal progenitor cells, and their differentiation is affected by genetics, maternal nutrition, and physiological status. Adipogenesis in beef cattle initiates first in visceral fat around mid gestation followed by subcutaneous, intermuscular, and intramuscular adipogenesis, which extends to approximately the neonatal stage for visceral fat and to early weaning stage for subcutaneous and intermuscular fat and to around 250 d of age for intramuscular adipogenesis. It is possible to specifically enhance intramuscular adipogenesis through nutrient supplementation targeted to a specific developmental stage, that is, from early weaning to 250 d or the so-called marbling window. Our understanding about mechanisms regulating progenitor cell differentiation into myogenic, adipogenic, and fibrogenic cells remain rudimentary. Elucidation of such mechanisms will make it possible to manipulate progenitor cell differentiation, maximizing production efficiency and quality of beef production. LITERATURE CITED Aberle, E. D Myofiber differentiation in skeletal muscles of newborn runt and normal weight pigs. J. Anim. Sci. 59: Ailhaud, G Adipose tissue as a secretory organ: From adipogenesis to the metabolic syndrome. C. R. Biol. 329: Alessi, M. C., F. Peiretti, P. Morange, M. Henry, G. Nalbone, and I. Juhan-Vague Production of plasminogen activator inhibitor 1 by human adipose tissue: Possible link between visceral fat accumulation and vascular disease. 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