Biomedical subjects

Federica Montanaro

Publications and source records attributed to Federica Montanaro.

4 recordsLinked to original sources

PubMed2004

Delta-sarcoglycan is required for early zebrafish muscle organization.

Mutations in sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-sarcoglycan primarily show adult phenotypes, our results suggest that delta-sarcoglycan plays a role in early zebrafish muscle development.

Amino Acid Sequence↗

PubMed2004

Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters.

Side population (SP) cells isolated from bone marrow, skeletal muscle, and skin have been shown to engraft in dystrophic muscle. However, there have been questions on the phenotypical heterogeneity, tissue of origin, and relationships among SP cell populations extracted from different tissues. Studies on bone marrow SP cells have followed a consistent protocol for their isolation and results obtained are concordant. In contrast, protocols for the isolation of muscle SP cells vary greatly, and consequently reports on their phenotype, differentiation potential and origin have been inconsistent. To address this controversy, we demonstrate that isolation parameters, such as tissue dissociation, cell counting, Hoechst concentration, and stringency in the selection of SP cells, have an effect on the yield, viability, and homogeneity of SP cells derived from bone marrow, skeletal muscle, and skin. In this paper, we demonstrate that SP cells isolated from the bone marrow are distinct from SP cells extracted from skeletal muscle and skin tissues. This study offers an explanation for the controversy surrounding muscle SP cells, provides a detailed standardized protocol for their isolation, and highlights basic guidelines for reproducible and reliable isolation of SP cells from any tissue.

Animals↗

PubMed2003

Expression profiling and identification of novel genes involved in myogenic differentiation.

Skeletal muscle differentiation is a complex, highly coordinated process that relies on precise temporal gene expression patterns. To better understand this cascade of transcriptional events, we used expression profiling to analyze gene expression in a 12-day time course of differentiating C2C12 myoblasts. Cluster analysis specific for time-ordered microarray experiments classified 2895 genes and ESTs with variable expression levels between proliferating and differentiating cells into 22 clusters with distinct expression patterns during myogenesis. Expression patterns for several known and novel genes were independently confirmed by real-time quantitative RT-PCR and/or Western blotting and immunofluorescence. MyoD and MEF family members exhibited unique expression kinetics that were highly coordinated with cell-cycle withdrawal regulators. Among genes with peak expression levels during cell cycle withdrawal were Vcam1, Itgb3, Itga5, Vcl, as well as Ptger4, a gene not previously associated with the process of myogenesis. One interesting uncharacterized transcript that is highly induced during myogenesis encodes several immunoglobulin repeats with sequence similarity to titin, a large sarcomeric protein. These data sets identify many additional uncharacterized transcripts that may play important functions in muscle cell proliferation and differentiation and provide a baseline for comparison with C2C12 cells expressing various mutant genes involved in myopathic disorders.

Animals↗

PubMed2003

Skeletal muscle engraftment potential of adult mouse skin side population cells.

Adult bone marrow and skeletal muscle have been shown to contain a subpopulation of cells, called side population (SP) cells, that can be isolated with the fluorescence-activated cell sorter. We used a similar method to identify SP cells in the skin of adult mice. These cells express surface markers similar to SP cells isolated from skeletal muscle, but differ from bone marrow SP cells and do not express hematopoietic markers. When transplanted into nonirradiated mdx mice, nuclei from donor skin SP cells are found within myofibers that express dystrophin. Thus, adult skin SP cells can engraft in dystrophic skeletal muscle even in the absence of total body irradiation.

Animals↗