Cell Lineage Trees in Mice Figure 2. Lineage reconstruction of ML2 and ML4. Reconstructed trees of 26 cells of ML2 (10 week old mouse) and 25 cells of ML4 (13 week old mouse) are shown. Terminal nodes represent single analyzed cells (colors are used for different cell types), while intermediate nodes (uncolored) represent hypothesized precursor cells. Reconstruction was performed using the Neighbor-Joining algorithm (with the distance function ‘Absolute- distance’, see Methods). The vertical axis represents number of cell divisions, and the estimated depth for each cell is obtained by projecting it to this axis. These estimates were obtained by a method we developed, which is based on the correlation between genetic distance and cell depth [15]. Reconstructed trees show that none of the analyzed cell types is completely and solely clustered on a subtree, demonstrating that none of cell types is a single exclusive clones. doi:10.1371/journal.pone.0001939.g002 in each of which reside about 10–20 satellite cells [16] (Figure 4E). This hierarchy between the various components, which together make skeletal muscle, led us to ask whether satellite cells from the same muscle have a significantly longer common developmental path than satellite cells from different muscles. Satellite cells have yet another advantage which enables performing this type of analysis–they can be isolated from pre-determined physical locations in the mouse while preserving this information for each analyzed cell (Figure 4A–D). In each experiment we isolated several satellite cells from various muscles and myofibers (see Table 1). We labeled each satellite cell according to its laterality (left or right), muscle identity (e.g. Gastrocnemius), and myofiber (e.g. 2nd myofiber of Gastrocnemius). We analyzed the correlation between length of common developmental history and myofiber, muscle and body- side, as described above (Figure 4F). We found that satellite cells from the same myofiber have a significantly longer common developmental history than a random pair of cells (p,0.0001) using both measures (MRCA depth and percent of common mutations). Satellite cells from the same muscle also have a significantly longer common developmental history than a random pair of cells (p,0.002 for both measures), but this is because of the significance of myofibers (satellite cells from the same myofiber are necessarily also from the same muscle). To test the significance of muscle independent of myofiber, we analyzed satellite cells pairs from the same muscle, but different myofibers. In this case, they did not have a significantly longer common developmental history than a random pair of cells (p.0.32 for both measures). Satellite cell pairs from the same body-side did not show a significantly longer common developmental history than a random pair of cells using any of the measures. Taken together, then results suggest that myofiber is a significant lineage unit in mouse. Physically Closer Satellite Cells Share a Longer Common Developmental History Next, we analyzed whether physically closer satellite cells share a longer common developmental history. Myofibers are numbered according to the order in which they were encountered when penetrating the muscle, from superficial to deepest. This implies that myofibers with adjacent indexes are also close physically. We define the physical distance between two myofibers to be the difference between their indices (e.g. the distance between two satellite cells from the same muscle, one from myofiber #3 and the other from myofiber #1, is 2). We analyzed the two main muscles in ML7: Gastrocnemius-Left (including 23 satellite cells from 4 myofibers) and Gastrocnemius-Right (23 satellite cells from 5 myofibers). The other two muscles (EDL-Left and EDL-Right) were comprised of a small number of satellite cells (3 and 8, PLoS ONE | www.plosone.org 4 April 2008 | Volume 3 | Issue 4 | e1939