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The regulation of myogenic progenitor cells during muscle regeneration is not clearly understood. We have previously shown that the Foxk1 gene, a member of the forkhead/winged helix family of transcription factors, is expressed in myogenic progenitor cells in adult skeletal muscle. In the present study, we utilize transgenic technology and demonstrate that the 4.6 kb upstream fragment of the Foxk1 gene directs beta-galactosidase expression to the myogenic progenitor cell population. We further establish that Sox15 directs Foxk1 expression to the myogenic progenitor cell population, as it binds to an evolutionarily conserved site and recruits Fhl3 to transcriptionally coactivate Foxk1 gene expression. Knockdown of endogenous Sox15 results in perturbed cell cycle kinetics and decreased Foxk1 expression. Furthermore, Sox15 mutant mice display perturbed skeletal muscle regeneration, due in part to decreased numbers of satellite cells and decreased Foxk1 expression. These studies demonstrate that Sox15, Fhl3 and Foxk1 function to coordinately regulate the myogenic progenitor cell population and skeletal muscle regeneration.

The successful use of myogenic progenitor cells for therapeutic applications requires an understanding of the intrinsic and extrinsic cues involved in their regulation. Herein we demonstrate the expression pattern and transcriptional regulation of Rad, a prototypical member of a family of novel Ras-related GTPases, during mammalian development and skeletal muscle regeneration. Rad was identified using microarray analysis, which revealed robust upregulation of its expression during skeletal muscle regeneration. Our current findings demonstrate negligible Rad expression with resting adult skeletal muscle; however, after muscle injury, Rad is expressed within the myogenic progenitor cell population. Rad expression is significantly increased and localized to the myogenic progenitor cell population during the early phases of regeneration and within the newly regenerated myofibers during the later phases of regeneration. Immunohistochemical analysis demonstrated that Rad and MyoD are coexpressed within the myogenic progenitor cell population of regenerating skeletal muscle. This expression profile of Rad during skeletal muscle regeneration is consistent with the proposed roles for Rad in the inhibition of L-type Ca(2+) channel activity and the inhibition of Rho/RhoA kinase activity. We also have demonstrated that known myogenic transcription factors (MEF2, MyoD, and Myf-5) can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by the presence of the Ca(2+)-dependent phosphatase calcineurin. Furthermore, this enhanced transcriptional activity appears to be dependent on the presence of a conserved NFAT binding motif within the Rad promoter. Taken together, these data define Rad as a novel factor within the myogenic progenitor cells of skeletal muscle and identify key regulators of its transcriptional activity.

Stem cells are important in the maintenance and repair of adult tissues. A population of cells, termed side population (SP) cells, has stem cell characteristics as they have been shown to contribute to diverse lineages. In this study, we confirm that Abcg2 is a determinant of the SP cell phenotype. Therefore, we examined Abcg2 expression during murine embryogenesis and observed robust expression in the blood islands of the E8.5 yolk sac and in developing tissues including the heart. During the latter stages of embryogenesis, Abcg2 identifies a rare cell population in the developing organs. We further establish that the adult heart contains an Abcg2 expressing SP cell population and these progenitor cells are capable of proliferation and differentiation. We define the molecular signature of cardiac SP cells and compare it to embryonic stem cells and adult cardiomyocytes using emerging technologies. We propose that the cardiac SP cell population functions as a progenitor cell population for the development, maintenance, and repair of the heart.

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>6-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function. [ABSTRACT FROM AUTHOR]