Skeletal Muscle Neural Precursor Figure 1. Nestin-GFP+ cells from FDB muscle in culture. A. Cells were grown for 14 days and fixed at various times (1, 4, 7 and 14 days). Nestin- GFP, Tuj1, and Hoechst 33342 were merged with phase contrast. Scale bar = 100 mm. B. Relative proportion of Tuj1+ and MyoD+ cells at different times in the nestin-GFP+ population. Notice the disappearance of MyoD expression at day 5 and the progressive increase in Tuj1+ cells to become almost the only population by days 7 and 14 in culture. doi:10.1371/journal.pone.0016816.g001 which indicates that nestin expression at this stage fails to differentiate between the two cell progenitors. MyoD+ cells do not react to Tuj1 antibody and vice versa in more than 1,000 cells from 8 FDB muscles, supporting the concept that these two markers clearly identify two distinct cell populations: one committed to muscle and the other to neural lineages. At day 3, neural outpaced myogenic precursor cells to become the predominant or almost exclusive population in nestin-GFP+ cells. Nestin-GFP+/MyoD+ cells gradually began to decrease, representing 9.563.2%, while Tuj1+ cells were already 40.468% of all nestin-GFP+ cells. One factor contributing to this predominance is the decline of nestin-GFP expression in muscle progenitor cells with time in culture [25]. As early as day 4, almost three-fourths of nestin-GFP+ cells (73.864.8%) were Tuj1+, while nestin-GFP+/MyoD+ cells represented only 2.762.7%. This finding is consistent with previous reports of GFP fluorescence disappearing in satellite cells that are MyoD+ at a similar time point [25]. PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e16816