Abstract
Abstract: :
Purpose: Although extraocular muscle (EOM) is fundamentally distinct from other skeletal muscles, little is known of the developmental mechanisms underlying their divergence. Here, we developed EOM and hindlimb muscle cell lines and used these to evaluate early stages of in vitro myogenesis by morphologic and gene expression profiling techniques. Methods: Immortalized myoblast cell lines were derived from EOM (mEOM) and hindlimb muscles (mLM) of neonatal C57Bl/10 mice. After expansion of myoblasts, myogenesis was induced by transient reduction in media serum concentration. Myogenesis in the two cell lines was evaluated by morphology and gene expression profiling (Affymetrix mouse 430A & B arrays) at 5 intervals from 0 to 48 hours after induction. Several bioinformatic tools, including RMA, CAGED, and SOM, were used to identify differentially expressed transcripts. Results: Primary myoblast cultures spontaneously transformed after 7–12 passages and the resultant mEOM and mLM cell lines are maintained in our lab. Both myoblast lines expressed traditional myogenic regulatory factors and myoblasts fused to form myotubes within 24 hrs of induction. mEOM myoblasts/myotubes were consistently smaller than in mLM. Microarray analysis revealed substantial baseline differences between the myoblast lines and in the myogenesis of the two lines. mEOM and mLM myoblasts exhibited baseline (pre–induction) differences in > 500 transcripts. Moreover, the mEOM and mLM lines had 279 and 509 transcripts, respectively, dynamically modulated (≥ 2–fold change) during the first 48 hrs of myogenesis, only 125 of which were conserved across the two cell lines. The vast majority of mEOM–specific or mEOM–enriched transcripts were not traditional muscle–specific genes, but instead included a broad range of functional classes. Conclusions: The absence of an adequate in vitro model of EOM myogenesis has severely hampered mechanistic studies of the developmental regulation of its novel phenotype. Here, we established immortalized cell lines as key tools for study of early EOM myogenesis. Gene expression profiles suggest that EOM myoblasts are fundamentally different from those of other skeletal muscles and that early myogenic mechanisms exhibit a significant degree of muscle group specificity. Collectively, these and other data support the hypothesis that the EOM divergence from prototypical skeletal muscle is a consequence of both myoblast cell lineage and differential response to extrinsic (e.g., innervation patterns, load) influences.
Keywords: extraocular muscles: development • gene microarray