Laser microdissection-based expression analysis of key genes involved in muscle regeneration in mdx mice.
We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis-regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin-Eosin staining, different types of degenerative-regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3-5days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFbeta-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5-7 days post-necrosis). mdx degenerative-regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.
Marotta
Mario
M
Laboratori de Neurología Infantil, Institut de Recerca Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain. mmarotta@ir.vhebron.net
Sarria
Yaris
Y
Ruiz-Roig
Claudia
C
Munell
Francina
F
Roig-Quilis
Manuel
M
D000818:Animals; D005786:Gene Expression Regulation / Q000502:physiology*; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D042282:Microdissection / Q000379:methods; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D009137:Muscular Dystrophy, Animal / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D017570:MyoD Protein; D051787:Myogenic Regulatory Factor 5; D018008:Myogenin; D012333:RNA, Messenger / Q000096:biosynthesis; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D009124:Muscle Proteins; C497418:Myf5 protein, mouse; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D051787:Myogenic Regulatory Factor 5; D018008:Myogenin; D012333:RNA, Messenger