Progress 09/01/00 to 01/31/05
Outputs
The long-term goals of this project were to understand the mechanisms by which muscles first form in birds. We focused on understanding myogenesis within the somite of the chicken and quail with special emphasis on the myotome within the somite, the first site of skeletal muscle fibers form and on when satellite cells first appear. This work has important implications for the formation of muscles of differing types that could result in improved production efficiency of chicken and quail muscle in conventional breeding programs. During embryonic development cells become committed to form muscle fibers of different types and the type of each anatomic muscle in the bird is established in the embryo. The following contributions were made with support of this grant: 1) There is an intrinsic commitment to fiber-type on the part of the myoblast found in the somite. Commitment within the somite is independent of extrinsic signals such as innervation or signaling molecules
provided by the mesenchymal stroma in which muscles differentiate. Therefore, myoblasts formed in the somite determine the fiber type that they will produce when they migrate into the wing. 2) FGF8, a signaling molecule, regulates both myogenesis and chondrogenesis within the somite and its expression is under the control of sonic hedgehog. We reveal its role in muscle and rib formation in the chicken. 3) The temporal appearance and spatial distribution of fast and slow fiber types within the somite has a precise pattern. Fast fibers form first followed within hours by slow myosin expressing fibers. Most of fiber patterning in the somite is independent of innervation by motor nerves; only the later appearance of slow MyHC 2 requires innervation. Sonic-hedgehog increases the accumulation of both fast and slow fibers and fiber size in the somite, but has no effect on myotomal fiber phenotype. Models of formation muscle of the back must account for the positioning of the oldest fibers in
the ventral-lateral region of the myotome and the youngest fibers in the dorsomedial region. 4) Expression of Pax-7, N-CAM, cMet, M-Cadherin are widely accepted markers of satellite cells in adult muscle, and Pax-7 is proposed to be involved in satellite cell formation. We determined when these markers first appear within chicken muscles. We found Pax-7 is expressed at all stages of development In vivo from early embryonic day 5 through adult. Pax-7-expression disappears with myoblast cell fusion and is expressed in an oscillating pattern with sequential rounds of myoblast proliferation and fusion. Clonal daughters of a single myoblast can be either Pax-7 positive or negative. While the identification of Pax7-positive cells in the early limb could be interpreted as evidence that the satellite cell lineage is present contemporaneous with the embryonic and fetal myogenic lineages, we think that Pax7 is more likely to be a marker of cell commitment to any of the three known myogenic
lineages. We conclude that Pax-7 is a marker of committment to the myogenic lineage at every stage of muscle development.
Impacts
It is during earlier embryonic period (within the somite) that the subsequent patterns and extent of muscle formation in the chicken is determined. Therefore, by understanding how fibers of differing types are laid down in the somite, there is the prospect of increased efficiency of poultry meat production and controlling the type of muscle that forms. There may be the possibility of altering white and red meat composition of chickens by understanding these processes. It is known that broilers have more rapid growth and greater muscularity from rapid hypertrophy of the more numerous fast muscle fibers than occurs within the layer. Fast growing strains of chickens appear to have more muscle fibers of both fast and slow types when compared with those chickens of slower growth rate. Understanding factors that can alter muscle fiber types and fiber growth could provide a genetic basis for control of meat production.
Publications
- Stockdale FE, Nikovits W Jr, Christ B. Molecular and cellular biology of avian somite development. Dev Dyn. 219:304-21, 2000
- Nikovits, Jr., W., G.Cann, R. Huang, B. Christ, F.E. Stockdale. Patterning of fast and slow fibers with embryonic muscles is established independently of signals from the surrounding mesenchyme. Development, 128:2537-2544, 2001.
- Patel, K., B. Christ, and F.E. Stockdale. Control of muscle size during embryonic, fetal, and adult life. In: Vertebrate Myogenesis-Results and Problems in Cell Differentiation. Ed. B. Brand-Saberi. Pub. Springer. Pp 163-186, 2002.
- Stockdale, F.E, W. Nikovits, Jr., and N. Espinoza. Slow myosins in muscle development. In: Vertebrate Myogenesis-Results and Problems in Cell Differentiation. Ed. B. Brand-Saberi. Pub. Springer. Pp 199-214, 2002.
- Huang, R., D. Stolte, H. Kurz, F. Ehehalt, G.M. Cann, F.E. Stockdale, K. Patel, and B. Christ. Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development. Dev. Biol. 255:30-47, 2003.
- Sacks, L., G.M. Cann, W. Nikovits, Jr., S. Conlon, N. Espinoza, and F.E. Stockdale. Regulation of myosin expression during myotome formation. Development 130:3391-3402, 2003.
- Nikovits, Jr. W., S. Conlon, C. Noack, L. Sacks and F. E. Stockdale Pax-7 Expression in Satellite Cells and the Developmental Origin of Satellite Cells. 2005 (In preparation).
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Progress 10/01/02 to 09/30/03
Outputs
This Progress Report covers accomplishments of the first 12 months of this funding period. The long-term goals of this project are to understand the mechanisms by which muscles form in birds. We have focused on understanding of myogenesis within the somite of the chicken and quail with special emphasis on the myotome, the earliest site of skeletal muscle formation. This work has important implications for the formation of muscles of differing functional types that could improved production efficiency of chicken and quail muscle in conventional breeding programs. During embryonic development cells become committed to form muscle fibers of varying types and the fiber type composition of each anatomic muscle is established. In the chicken, the entire musculature below the head derives from the myotome of the somite, regularly iterated blocks of mesodermal cells that form adjacent to, and under the influence, of the neural tube and notochord (Stockdale et al., 2000). In
this period we have investigated the appearance of the three avian isoforms of slow myosin (MyHC) during formation and maturation of the myotome in the chicken. We showed by whole mount in situ hybridization (ISH), immunohistochemistry (IHC), and RT-PCR analyses using isoform-specific probes, the embryonic fast MyHC (efast MyHC) gene and all three slow MyHC genes are expressed in myotomal fibers. From the onset of expression, mRNA transcripts from the efast MyHC gene are distributed throughout the cytoplasm of myotomal fibers while the mRNA transcripts for all three slow MyHC family members are restricted to the central, nuclear domain. We investigated the mechanism(s) regulating the appearance of the various MyHC isoforms using surgical and pharmacological methods coupled with ISH and IHC. We found by PCR analysis and in situ hybridization that there are differences in the temporal appearance and spatial distribution of slow and fast myosin heavy chain mRNA transcripts within
myotomal fibers. Embryonic fast MyHC was the first isoform expressed, followed rapidly by slow MyHCs 1 and 3, with slow MyHC 2 appearing several hours later. As development proceeds, slow MyHC transcripts spread throughout the fibers of the myotome, in the order of their appearance. When individual or small groups of somites are removed from the embryo and incubated in organ culture with neural tube, notochord, they become innervated by motor neurons from the neural tube and express all 4 MyHC genes. Removal of the neural tube/notochord from somite explants prior to incubation or addition of d-tubocurare to intact explants prevented expression of slow MyHC 2 but expression of the other MyHC isoforms was unaffected. Thus expression of slow MyHC 2 is dependent on functional innervation, whereas expression of embryonic fast, and slow MyHCs 1 and 3 are innervation-independent in the first muscle fiber to form in the trunk of the chick and quail embryo. Differentiation in the myotome
proceeded first ventro-laterally and finally dorso-medially. These observations have implications for the proposed mechanisms of myotome formation.
Impacts
It is during the period of myotome formation the subsequent patterns and extent of muscle formation in the chicken is determined. Therefore, by understanding how fibers of differing types are laid down in the myotomes, there is the prospect of increased efficiency of poultry meat production. There may also be the possibility of altering white and red meat composition of chickens. For example, studies indicate that broilers have more muscle fibers than do layers. The broilers have more rapid growth and greater muscularity which appears to be from a rapid hypertrophy of the more numerous muscle fibers in the broiler compared to the layer. Fast growing strains of chickens appear to have more muscle fibers of both fast and slow types when compared with those of slower growth rate. Thus understanding factors that can alter muscle fiber types and fiber growth could provide a genetic basis for control of meat production.
Publications
- Patel, K., B. Christ, and F.E. Stockdale. Control of muscle size during embryonic, fetal, and adult life. In: Vertebrate Myogenesis-Results and Problems in Cell Differentiation. Ed. B. Brand-Saberi. Pub. Springer. Pp 163-186, 2002.
- Stockdale, F.E, W. Nikovits, Jr., and N. Espinoza. Slow myosins in muscle development. In: Vertebrate Myogenesis-Results and Problems in Cell Differentiation. Ed. B. Brand-Saberi. Pub. Springer. Pp 199-214, 2002.
- Huang, R., D. Stolte, H. Kurz, F. Ehehalt, G.M. Cann, F.E. Stockdale, K. Patel, and B. Christ. Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development. Dev. Biol. 255:30-47, 2003.
- Sacks, L., G.M. Cann, W. Nikovits, Jr., S. Conlon, N. Espinoza, and F.E. Stockdale. Regulation of myosin expression during myotome formation. Development 130:3391-3402, 2003.
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