Source: STANFORD UNIV submitted to
GROWTH & DEVELOPMENT OF CHICKEN MUSCLES
 
PROJECT DIRECTOR: Stockdale, F. E.
 
PERFORMING ORGANIZATION
dept of medicine/oncology
STANFORD UNIV
STANFORD,CA 94305
 
NON TECHNICAL SUMMARY: Our goal is to understand the mechanisms responsible for the formation of diverse muscle types in the developing chicken. We know that all muscles form from the tissues adjacent to the nervous system in the chick embryo. The tissue from which they form is called the myotome -the first muscle to form and the source from which virtually every muscle of the adult bird is derived. Our ultimate objectives are to understand how precursor cells form myotomal muscle fibers of different functional types. The types of contractile proteins they contain -often distinguished as 'red' or 'white' meat~ determine the function of muscles. It is during the period of myotome formation that the subsequent pattern of these two basic types of muscles is determined. Our f1!St aim is to analyze the influence of the developing nervous system, and signaling molecules emanating from the nervous system on expression of genes that control the type of muscles tl1at will form. We postulate that signals from the nervous system and the nerves that connect the spinal cord with muscles, regulate formation of the myotome and provide instructional information for myotome formation and contractile muscle protein gene expression. Our second aim is to examine the pathways that transmit these signals to the forming muscle cells to instruct their genes to become active. By understanding how fibers of differing types are laid down in the chicken (and quail), there is the prospect of increased efficiency of poultry meat production and alerting white and red meat composition of chickens.
 
OBJECTIVES: To understand the mechanisms by which muscles first form in birds. Understand the cellular interactions and molecules responsible for initiating fiber type diversity within myotomal muscle. Evaluate the hypothesis that signaling molecules from the neural tube and notochord initiate the differentiation of muscle cell precursors to form nascent fibers that exhibit innervation-independent and -dependent differentiation mechanisms and that axons emerging from the neural tube control maturation of myotomal fiber types by modulating slow and fast MyHC gene expression. Analyze the influence of the developjng neural tube, motor neurons, and signaling molecules emanating from the neural tube/notochord on fast and slow MyHC gene expression and muscle fiber fom1ation within the myotome of the chick embryo. Examine the role of various signal transduction pathways in the regulation of muscle fiber type in the myotome and epaxial musculature. Determine the cellular distribution of sequentially appearing slow MyHC mRNAs; the sequential appearance of slow MyHC protein in the myotome, and the role of innervation in myotomal fiber differentiation. Investigate how intercellular calcium pathways operating through calcineurin and protein kinases A and C, affect muscle fiber type and influence the expression of fiber type-specific MyHC genes.
 
APPROACH: In situ hybridization, irnmunohistochemistry and embryonic microsurgery in ovo, will be used determine if interference with motor neuron formation. neuromuscular transmission, or proximity of axon outgrowth from the nervous system affects the sequential expression of MyHCs in myotomal fibers of the chicken and the quail. A somite explant culture system, immunohistochemical staining for slow and fast MyHCs. and in situ hybridization with probes for specific myosin mRNA. and transfections of myogenic cells in vitro and in vivo to investigate signal transduction pathways will be used. Calcineurin pathway(s) in developing myotomes will be disrupted using retroviruses to deliver inhibitors specific for NFAT and calcineurin. Inhibitors of protein kinase A and protein kinase C will be used to disrupt signals operating through these kinases, Protein kinase C action on differentiation will be investigated to somites in culture using transfection techniques. These approaches will provide insights into how interaction between axonal outgrowth from the neural tube can affect fiber type via intracellular calcium dependent mechanisms and other signal ransduction pathways.
 
CRIS NUMBER: 0193649 SUBFILE: CRIS
PROJECT NUMBER: CALR-2002-03613 SPONSOR AGENCY: CSREES
PROJECT TYPE: NRI COMPETITIVE GRANT PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Sep 1, 2000 TERMINATION DATE: Jan 31, 2005

GRANT PROGRAM: IMPROVING ANIMAL GROWTH AND DEVELOPMENT
GRANT PROGRAM AREA: Animal Systems

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
305322010402.2100%

CLASSIFICATION HEADINGS
KA305 - Animal Physiological Processes
S3220 - Meat-type chicken, live animal
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 100%
APPLIED (N/A)%
DEVELOPMENTAL (N/A)%

KEYWORDS: muscles; chickens; animal development; animal growth; quail; muscle fibers; animal physiology; molecular biology; broilers; embryos; myosins; genes; protein kinase; nervous system; mechanism of action; muscle development; muscle growth; muscle proteins; differentiation; embryo development; axons; gene expression; signal transduction; metabolic pathways; myoblasts; calcium metabolism; poultry meat

PROGRESS: Sep 1, 2000 TO Jan 31, 2005
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.

IMPACT: 2000-09-01 TO 2005-01-31 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.

PUBLICATION INFORMATION: 2000-09-01 TO 2005-01-31
Stockdale FE, Nikovits W Jr, Christ B. Molecular and cellular biology of avian somite development. Dev Dyn. 219:304-21, 2000

PROJECT CONTACT INFORMATION
NAME: Stockdale, F. E.
PHONE: 650-725-6449
FAX: 650-736-2282