Progress 10/01/04 to 09/30/05
Outputs
The relationships between the expression of the myogenic regulatory factors Myf5, MyoD and myogenin and the paired box transcription factor Pax7 has been investigated in conjunction with cell proliferation. As previously reported, we demonstrated through double immunostaining of primary myoblasts isolated from posthatch chickens that Pax7+/MyoD+ cells likely represent the proliferative compartment, while Pax7+/MyoD- may represent reserve cells that will go on to reenter a quiescent state as a renewed satellite cell population. Pax7-/MyoD+ cells represent cells that have differentiated and have begun to express myogenin. While most myogenin+ cells are negative for Pax7, some myogenin+ cells do express Pax7 and likely represent cells transiting from proliferation to differentiation. It has also been observed through double immunostaining that all Myf5+ cells coexpress Pax7, but Pax7+ cells do not always express Myf5. The observed Pax7+/MyoD- cells could not be merely
Pax7+ cells that express Myf5 and not MyoD because the number of Myf5+ cells declined drastically with time in culture to levels far below that of the observed Pax7+/MyoD- cells. Hence, we conclude that some Pax7+ cells in chicken myogenic cultures do not express MyoD or Myf5. The different cell phenotypes have now been investigated in combination with bromodeoxyuridine (BrdU) labeling in chicken myogenic cultures grown to different stages in order to determine their proliferative capacity. Triple immunostaining for BrdU, Pax7, in combination with MyoD or Myf5 or myogenin, revealed that during early days in culture (i.e., day 4) almost all BrdU+ cells are Pax7+/MyoD+ or Pax7+/Myf5+ but negative for myogenin. At a later time point (i.e., day 10) when numerous cells have fused into myotubes, there are still proliferating (BrdU+) cells expressing Pax7. However, the majority of these cells were negative for Myf5 and myogenin while about two thirds of these BrdU+/Pax7+ cells were positive
for MyoD. Hence, this study identifies a population of BrdU+/Pax7+ cells that is negative for all three myogenic transcription factors. Collectively, Pax7+ cells in S phase (BrdU+) are heterogeneous with regard to Myf5 and MyoD expression, and there are some proliferating cells that do not express Myf5 and MyoD, the two myogenic transcription factors that characterize proliferating myoblasts. These proliferating Pax7+ cells (that are negative for the myogenic transcription factors) may represent a path toward replenishment of quiescent, Pax7+ cells (presumably satellite cells). The cell population dynamics described above are being further examined in chicken myogenic cultures maintained in serum-reduced and serum-free media for optimizing conditions for studying the effect of growth factors as well as pharmacological inhibitors of several tyrosine kinase receptors of relevance to satellite cell proliferation and differentiation.
Impacts
Through investigating the various compartments using triple immunostaining for Pax7, BrdU and myogenic regulatory factors, we have been able to further define these compartments and to isolate the potential pool of proliferating cells that are determined to become the next generation of satellite cells. The working model we have established permit us to investigate the effect of various growth factors on the mechanism of satellite cell proliferation, differentiation and self-renewal. Future utilization of our finding may enhance the efficiency of meat production by shortening the time required for poultry to reach market size.
Publications
- Shefer G, Yablonka-Reuveni Z. 2005 Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol. Biol. 290:281-304.
- Yablonka-Reuveni Z, Anderson JE. 2006 Satellite cells from dystrophic (Mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers. Dev. Dyn. 235: 203-212.
- Halevy O, Piestun Y, Rozenboim I, Yablonka-Reuveni Z. In-ovo exposure to monochromatic green light promotes skeletal muscle cell proliferation and affects myofiber growth in posthatch chicks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005 Nov 3; [Epub ahead of print]
- Shefer G, Van de Mark DP, Richardson JB, Yablonka-Reuveni Z. 2005 Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle. Submitted.
- Allouh M, Yablonka-Reuveni Z, Rosser BWC. 2006 Distribution of satellite cells along the myofiber length in growing and adult chicken. Submitted.
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Progress 10/01/03 to 09/30/04
Outputs
Myoblasts, isolated from posthatch chickens and cultured under routine conditions (primary cultures), were investigated for the expression of Pax7 and MyoD or Pax7 and Myf5 using double immunostaining. When cultures were reacted with antibodies to Pax7 and MyoD, both single-stained (Pax7+/MyoD-, Pax7-/MyoD+) and double-stained (Pax7+/MyoD+) cells were identified; as we previously shown, Pax7+/MyoD- cells likely represent the reserve cell compartment, Pax7+/MyoD+ cells represent the proliferative compartment and Pax7-/MyoD+ cells reflect cells that have transited into the differentiated, myogenin+ compartment. Additionally we demonstrated by Western blotting and gel mobility shift that MyoD protein expressed by proliferating cells is phosphorylated but that expressed in differentiating cells is not. When cultures, parallel to those described above, were reacted with antibodies to Pax7 and Myf5, all Myf5+ cells were also positive for Pax7 but there were many Pax7+ cells
that expressed little, if any Myf5. The nuclear morphology of these Pax7+/MyF5- cells suggested that they are potentially in the M phase of the cell cycle. This has been further verified using double immunostaining with an antibody against phospho histone 3 (a mitotic marker). Furthermore, the persistent co-expression of Myf5 and Pax7 indicates that Myf5 expression, like Pax7 expression, is terminated upon differentiation. We further asked if Myf5 is expressed by quiescent satellite cells (like Pax7; we previously demonstrated that MyoD is not expressed by quiescent satellite cells). Myofibers were isolated from mice expressing nuclear lacZ under the control of the Myf5 promoter. Indeed, many of the satellite cells were found positive for lacZ as well as their proliferative progeny. Preliminary results suggest that different from Myf5 protein, lacZ expressed under the control of the Myf5 promoter continues to be expressed during the M phase. Clonal analysis of myoblasts from posthatch
chickens further indicated that all cell phenotypes seen in primary cultures can be found within each individual clones and are thus, lineally related. While the quiescent satellite cells express Pax7 (and possibly Myf5), the proliferative myobast can be characterized by the triple expression of pax7, MyoD and Myf5. Upon differentiation into the myogenin-expressing state, Pax7 and Myf5 expression terminates. The aforementioned studies refined our knowledge on the kinetics of muscle gene expression with relationships to proliferation and differentiation. This information is now beign used by us to evaluate the role of growth factors during the transitions of the cells through the multi-step program of myogenesis in the posthatch chicken.
Impacts
Our studies indicate that the expression of the three early myogenic markers: Pax7, MyoD and Myf5, follows different patterns. Proliferating cells express Pax7, Myf5 and MyoD but during mitosis Myf5 protein expression seems to decline. Expression of Pax7 and Myf5 declines when cells enter myogenin expression (i.e., onset of differentiation) but MyoD continues to be expressed. MyoD protein expressed by proliferating cells is phosphorylated but that expressed in differentiating cells is not. An ability to control the size of the pool of proliferating myoblsts may allow better synchronization of posthatch muscle growth and consequently enhance meat production efficiency.
Publications
- Yablonka-Reuveni, Z. 2004 Isolation and culture of myogenic stem cells. In: Handbook of Stem Cells, Vol 2 - Adult and Fetal Stem Cells. R. Lanza, H. Blau, D. Melton, M. Moore, E.D. Thomas, C. Verfaillie, I. Weissman and M. West. Eds. 571-580. Elsevier: Academic Press, San Diego.
- Galli, L.M., Willert, K., Nusse, R., Yablonka-Reuveni, Z., Nohno, T., Denetclaw, W., and Burrus, L.W. 2004 A Proliferative Role for Wnt-3a in Chick Somites. Dev. Biol. 269: 489-504
- Halevy O., Piestun Y., Allouh, M., Rosser, B.W.C., Rinkevich, Y., Reshef, R., Rozenboim, I., Wleklinski-Lee, M., and Yablonka-Reuveni, Z. 2004 The pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal. Dev. Dynamics. 231: 489-502
- Shefer, G., Wleklinski-Lee, M., and Yablonka-Reuveni, Z. 2004 Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway. J Cell Sci. 117: 5393-5404
- Shefer, G., and Yablonka-Reuveni, Z. 2004 Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol. 290:281-304
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Progress 10/01/02 to 09/30/03
Outputs
Myoblasts, isolated from 9-day posthatch chickens and cultured under routine conditions (primary cultures), were investigated for the expression of Pax7, MyoD and myogenin using double immunostaining. When cultures were reacted with antibodies to Pax7 and MyoD, both single-stained and double-stained cells were identified. Likewise, when the cultures were reacted with antibodies to Pax7 and myogenin, single-stained and double-stained cells were identified. At all time points analyzed the majority of myogenin+ cells were negative for Pax7. The time course of the different cell phenotypes additionally showed that in late cultures (i.e., 7-15 days following culture establishment) the number of Pax7+/MyoD- is greatly increased compared to early days in culture. Collectively, the kinetics observed support our initial hypothesis regarding the phenotypic path of the satellite cells as they exit the reserve cell compartment and transit through proliferation and
differentiation. This multi-compartment path should be however updated as follows to allow for a small number of Pax7+/Myogenin+ cells which we propose to be representative of an intermediate (and short lived) phase at the onset of differentiation: Pax7+/MyoD-/Myogenin- ---> Pax7+/MyoD+/Myogenin- ---> Pax7-MyoD+/Myogenin+ ---> Pax7+MyoD+/Myogenin+ ---> MyoD+/Myogenin+. The level of Pax7 immunosignal in the myogenin+ cells is typically low; the identification of the Pax7+/Myogenin+ cells has become possible after our initial conditions for fixing cultures for immunostaining have been modified. Importantly, the increased number of Pax7+ cells in the late cultures has suggested that, in concert with the transition of the proliferating myoblasts into differentiation, the proliferating cells also produce reserve myoblasts. Parallel studies were conducted with clonal cultures where each clone represents progeny of one myogenic precursor. The goal of the clonal studies was to verify if a
common myogenic progenitor gives rise to both proliferating (Pax7+/MyoD+) and differentiating myoblasts (Pax7-/myogenin+) cells, along with generating reserve cells (Pax7+/MyoD-). Alternatively, some progenitors may give rise only to the reserve cells (by proliferation of Pax7+/MyoD- cells) while others may produce only the differentiation-bound cells (Pax7+/MyoD+/Myogenin- and Pax7-/MyoD+/Myogenin+ cells). All clonal cultures contained both the reserve cells and the differentiation-bound cells. Hence, the clonal analysis supports our hypothesis that committed myoblasts are capable of differentiation and self renewal, potentially via asymmetric cell division. The studies with the primary culture have also provided important background information for our future investigation regarding the effect of growth factors on the transition of satellite cells from the Pax7+/MyoD-/Myogenin- state (reserve cells) to the Pax7+/MyoD+/Myogenin- state (proliferating cells). Cultures will be
maintained for up to two weeks to allow generation of many Pax7+/MyoD- cells. These cultures will then be used as Time 0 cultures to investigate the transition of the reserve cells into the proliferating compartment.
Impacts
Our in vivo studies indicate that Pax7+ cells represent the skeletal muscle satellite cells in posthatch and adult muscle. Our cell culture studies further suggest that Pax7+/MyoD-/Myogenin- cells represent the reserve myogenic pool in posthatch chickens. An ability to control the size of the reserve cell pool vs. the pool of proliferating myoblsts (Pax7+/MyoD+) and differentiating myoblasts (Pax7-/Myogenin+) may allow better synchronization of posthatch muscle growth and consequently enhance meat production efficiency.
Publications
- Galli, L.M., Willert, K., Nusse, R., Yablonka-Reuveni, Z., Nohno, T., Denetclaw, W., and Burrus, L.W. 2004 A Proliferative Role for Wnt-3a in Chick Somites. Submitted.
- Yablonka-Reuveni, Z. 2004 Isolation and culture of myogenic stem cells. In: Handbook of Stem Cells. Lanza, R.P., Blau, H.M., Melton, D.A., Moore, M.A.S., Thomas, E.D., Verfaillie, C.M., Weissman, I.L., and West, M.D., . Eds. Academic Press. In press.
- Halevy, O., Piestun, Y., Allouh, M.Z., Rosser, B., Rinkevich, Y., Reshef, R., Rozenboim, I., Wleklinski, M.,and Yablonka-Reuveni, Z. 2004 Pax7 expression during satellite cell myogenesis and muscle growth in posthatch chickens. Manuscript ready for submission.
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