Prediction of gene network models in limb muscle precursors.

The ventrolateral dermomyotome gives rise to all muscles of the limbs through the delamination and migration of cells into the limb buds. These cells proliferate and form myoblasts, withdraw from the cell cycle and become terminally differentiated. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Gene expression arrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1(EGFP), which resulted from the loss of Pitx2. Target genes of Pitx2 were clustered using the "David Bioinformatics Functional Annotation Tool" to bin genes according to enrichment of gene ontology keywords. This provided a way to both narrow the target genes and identify potential gene families regulated by Pitx2. Representative target genes in the most enriched bins were analyzed for the presence and evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the -20kb, intronic, and coding regions of the genes. Fifteen Pitx2 target genes were selected based on the above analysis and were identified as having functions involving cytoskeleton organization, tissue specification, and transcription factors. Data from these studies suggest that Pitx2 acts to regulate cell motility and expression of muscle specific genes in the muscle precursors during forelimb muscle development. This work provides a framework to develop the gene network leading to skeletal muscle development, growth and regeneration.