Wnt/ß-catenin controls follistatin signalling to regulate satellite cell myogenic potential.

BACKGROUND: Adult skeletal muscle regeneration is a highly orchestrated process involving the activation and proliferation of satellite cells, an adult skeletal muscle stem cell. Activated satellite cells generate a transient amplifying progenitor pool of myoblasts that commit to differentiation and fuse into multinucleated myotubes. During regeneration, canonical Wnt signalling is activated and has been implicated in regulating myogenic lineage progression and terminal differentiation. METHODS: Here, we have undertaken a gene expression analysis of committed satellite cell-derived myoblasts to examine their ability to respond to canonical Wnt/ß-catenin signalling. RESULTS: We found that activation of canonical Wnt signalling induces follistatin expression in myoblasts and promotes myoblast fusion in a follistatin-dependent manner. In growth conditions, canonical Wnt/ß-catenin signalling prime myoblasts for myogenic differentiation by stimulating myogenin and follistatin expression. We further found that myogenin binds elements in the follistatin promoter and thus acts downstream of myogenin during differentiation. Finally, ectopic activation of canonical Wnt signalling in vivo promoted premature differentiation during muscle regeneration following acute injury. CONCLUSIONS: Together, these data reveal a novel mechanism by which myogenin mediates the canonical Wnt/ß-catenin-dependent activation of follistatin and induction of the myogenic differentiation process.

Raman spectroscopy of uranium compounds and the use of multivariate analysis for visualization and classification.

Raman spectroscopy was used on 95 samples comprising mainly of uranium ore concentrates as well as some UF4 and UO2 samples, in order to classify uranium compounds for nuclear forensic purposes, for the first time. This technique was selected as it is non-destructive and rapid. The spectra obtained from 9 different classes of chemical compounds were subjected to multivariate data analysis such as principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and Fisher Discriminant Analysis (FDA). These classes were ammonium diuranate (ADU), sodium diuranate (SDU), ammonium uranyl carbonate (AUC), uranyl hydroxide (UH), UO2, UO3, UO4, U3O8 and UF4. Unsupervised PCA of full spectra shows fairly good distinction among the classes with some overlaps observed with ADU and UH. These overlaps are also reflected in the poorer specificities determined by PLS-DA. Higher values of sensitivities and specificities of remaining compounds were obtained. Supervised FDA based on reduced dataset of only 40 variables shows similar results to that of PCA but with closer clustering of ADU, UH, SDU, AUC. As a rapid and non-destructive technique, Raman spectroscopy is useful and complements existing techniques in multi-faceted nuclear forensics.

Classification of spent reactor fuel for nuclear forensics.

In this paper we demonstrate the use of pattern recognition and machine learning techniques to determine the reactor type from which spent reactor fuel has originated. This has been done using the isotopic and elemental measurements of the sample and proves to be very useful in the field of nuclear forensics. Nuclear materials contain many variables (impurities and isotopes) that are very difficult to consider individually. A method that considers all material parameters simultaneously is advantageous. Currently the field of nuclear forensics focuses on the analysis of key material properties to determine details about the materials processing history, for example, utilizing known half-lives of isotopes can determine when the material was last processed (Stanley, F. E. J. Anal. At. Spectrom. 2012, 27, 1821; Varga, Z.; Wallenius, M.; Mayer, K.; Keegan, E.; Millet, S. Anal. Chem. 2009, 81, 8327-8334). However, it has been demonstrated that multivariate statistical analysis of isotopic concentrations can complement these method and are able to make use of a greater level of information through dimensionality reduction techniques (Robel, M.; Kristo, M. J. J. Environ. Radioact. 2008, 99, 1789-1797; Robel, M.; Kristo, M. J.; Heller, M. A. Nuclear Forensic Inferences Using Iterative Multidimensional Statistics. In Proceedings of the Institute of Nuclear Materials Management 50th Annual Meeting, Tucson, AZ, July 2009; 12 pages; Nicolaou, G. J. Environ. Radioact. 2006, 86, 313-318; Pajo, L.; Mayer, K.; Koch, L. Fresenius' J. Anal. Chem. 2001, 371, 348-352). There has been some success in using such multidimensional statistical methods to determine details about the history of spent reactor fuel (Robel, M.; Kristo, M. J. J. Environ. Radioact. 2008, 99, 1789-1797). Here, we aim to expand on these findings by pursuing more robust dimensionality reduction techniques based on manifold embedding which are able to better capture the intrinsic data set information. Furthermore, we demonstrate the use of a number of classification algorithms to reliably determine the reactor type in which a spent fuel material has been irradiated. A number of these classification techniques are novel applications in nuclear forensics and expand on the existing knowledge in this field by creating a reliable and robust classification model. The results from this analysis show that our techniques have been very successful and further ascertain the excellent potential of these techniques in the field of nuclear forensics at least with regard to spent reactor fuel.

Pax7 is critical for the normal function of satellite cells in adult skeletal muscle.

Extensive analyses of mice carrying null mutations in paired box 7 (Pax7) have confirmed the progressive loss of the satellite cell lineage in skeletal muscle, resulting in severe muscle atrophy and death. A recent study using floxed alleles and tamoxifen-induced inactivation concluded that after 3 wk of age, Pax7 was entirely dispensable for satellite cell function. Here, we demonstrate that Pax7 is an absolute requirement for satellite cell function in adult skeletal muscle. Following Pax7 deletion, satellite cells and myoblasts exhibit cell-cycle arrest and dysregulation of myogenic regulatory factors. Maintenance of Pax7 deletion through continuous tamoxifen administration prevented regrowth of Pax7-expressing satellite cells and a profound muscle regeneration deficit that resembles the phenotype of skeletal muscle following genetically engineered ablation of satellite cells. Therefore, we conclude that Pax7 is essential for regulating the expansion and differentiation of satellite cells during both neonatal and adult myogenesis.

Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle.

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved (1). In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation (2, 3). The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions (3-6). The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation (7). Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles (8, 9) so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium (1-3). This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process (1-3). The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Transcriptional dominance of Pax7 in adult myogenesis is due to high-affinity recognition of homeodomain motifs.

Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development.

Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells.

Satellite cells in skeletal muscle are a heterogeneous population of stem cells and committed progenitors. We found that quiescent satellite stem cells expressed the Wnt receptor Fzd7 and that its candidate ligand Wnt7a was upregulated during regeneration. Wnt7a markedly stimulated the symmetric expansion of satellite stem cells but did not affect the growth or differentiation of myoblasts. Silencing of Fzd7 abrogated Wnt7a binding and stimulation of stem cell expansion. Wnt7a signaling induced the polarized distribution of the planar cell polarity effector Vangl2. Silencing of Vangl2 inhibited Wnt7a action on satellite stem cell expansion. Wnt7a overexpression enhanced muscle regeneration and increased both satellite cell numbers and the proportion of satellite stem cells. Muscle lacking Wnt7a exhibited a marked decrease in satellite cell number following regeneration. Therefore, Wnt7a signaling through the planar cell polarity pathway controls the homeostatic level of satellite stem cells and hence regulates the regenerative potential of muscle.

Transcriptional networks that regulate muscle stem cell function.

Muscle stem cells comprise different populations of stem and progenitor cells found in embryonic and adult tissues. A number of signaling and transcriptional networks are responsible for specification and survival of these cell populations and regulation of their behavior during growth and regeneration. Muscle progenitor cells are mostly derived from the somites of developing embryos, while satellite cells are the progenitor cells responsible for the majority of postnatal growth and adult muscle regeneration. In resting muscle, these stem cells are quiescent, but reenter the cell cycle during their activation, whereby they undergo decisions to self-renew, proliferate, or differentiate and fuse into multinucleated myofibers to repair damaged muscle. Regulation of muscle stem cell activity is under the precise control of a number of extrinsic signaling pathways and active transcriptional networks that dictate their behavior, fate, and regenerative potential. Here, we review the networks responsible for these different aspects of muscle stem cell biology and discuss prevalent parallels between mechanisms regulating the activity of embryonic muscle progenitor cells and adult satellite cells.

Successful treatment of pediatric stroke with recombinant tissue plasminogen activator (rt-PA): a case report and review of the literature.

Pediatric stroke is a rare disorder with a paucity of evidence-based treatment options, and can result in significant morbidity and mortality. In contrast to adult acute ischemic stroke, where the use of recombinant tissue plasminogen activator (rt-PA) has been studied in several large, randomized clinical trials, no high-level evidence exists for the use of thrombolytics in children with stroke. We report a case of a 15-year-old girl who suffered an acute ischemic stroke and had a dramatic improvement in symptoms following the administration of intravenous rt-PA.