While this is in accordance with the fact that cells in early embryogenesis retain a large developmental potential [20], to the best of our knowledge we are the first to demonstrate it in higher organisms. If any of the cells types were a single exclusive clone, we expect that analysis using our set of MS loci would be sufficient to detect it, since our group was able to detect clonality of tumor cells in distinct physical locations using a similar set of MS [Frumkin D. et al., submitted]. We note three special cases of single exclusive clones that are recognized in vertebrates: cancer, which according to the accepted dogma arises from a single founder cell [21], lymphocytes committed to the production of a particular species of antibody molecule, which arise as a single subclone from a pluripotent lymphocyte [9], and intestinal crypts, which are composed of a monoclonal population of cells descended from a single active multipotent stem cell [22]. The fact that we did not find a correlation between cell type and lineage may be a result of insufficient sampling. Specifically, to detect a significant lineage correlation in a cell population which is polyclonal, one needs to sample at least the number of progenitors of the population (Itzkovitz S. et al., manuscript in preparation). We can provide a lower bound and say that if cell populations such as B-cells sampled in this work arise from several independent clones, the number of clones is higher than the number of cells sampled. In our analysis, we focused on the length of the common developmental history of cells, as measured by MRCA depth in the reconstructed lineage tree and percent of common mutations. Other aspects of cell lineage can be compared as well, such as the path distance between cells in the reconstructed lineage tree. We did not present analysis results that use this measure since we found them to reflect mostly on the depth of cells. The second and third questions we addressed are related to the development and maintenance of body musculature. We asked whether there is a correlation between cell lineage and physical proximity in satellite cells within a muscle. We found a significant anti-correlation between physical proximity and the length of the common developmental path of these cells. This demonstrates that satellite cells from the same muscle which are physically closer to each other tend to share a longer common developmental path. Next, we asked whether there is a correlation between cell lineage and anatomical proximity among cells, based on the hierarchy of satellite cell-myofiber-muscle-body side. We found that satellite cells from the same myofiber are significantly clustered together on the lineage tree, suggesting they are created (and perhaps maintained) from a small pool of progenitor cells. Our data suggests that satellite cells from the same myofiber are not single exclusive clones because no myofiber is clustered on a distinct subtree. This means that satellite cells from a single myofiber are either created by more than one founder cell during embryogen- esis, or become heterogenic during the process of muscle repair following injury. These results may help understand the origins of satellite cells. Our data are most consistent with satellite cells being created locally and temporally during embryogenesis [23–25], rather than having a heterogenic origin from various tissues such as the bone marrow or other sources [26,27]. Our results are currently inconclusive regarding the correlation between muscle and cell lineage. On one hand, we found a significant correlation between physical proximity and depth of common ancestor within a muscle, suggesting that myofibers within muscles are also linked lineage-wise. On the other hand, satellite cells from the same muscle did not have longer common developmental history compared to satellite cells from different muscles. While these findings do not necessarily contradict, a conclusive statement in this respect requires further experiments with more muscles at various physical distances from each other (in ML7, which was our Cell Lineage Trees in Mice major experiment in satellite cells, we analyzed only two muscles, which were quite close physically). Regarding the correlation between cell lineage and body-side, our results demonstrate that there is no significant correlation between these, at least in satellite cells. In principle, it may be possible to reconstruct the entire satellite cell lineage, which would give a clearer picture regarding the development and maintenance of satellite cells, and should reveal their heterogenic nature. We have evidence that our method for reconstructing cell lineages from somatic mutations is informative and discovers relations between cells when these exist. First, we found that satellite cells from the same myofiber tend to share a longer common developmental path, which makes sense biologically. Second, our group analyzed cancerous and non-cancerous cells from an older mouse using our method and found that the tumor initiated from a single founder cell approximately five months prior to diagnosis and that the tumor grew in a physically coherent manner (Frumkin et al., submitted). These findings are in accordance with the accepted paradigm of cancer development. Third, our previous work [1] demonstrated an ex vivo proof of concept for the method. Extrapolation from in vitro to in vivo assumes that somatic mutations in MS occur similarly ex vivo and in vivo. Beyond the specific biological findings, we see the importance of this work as a demonstration of the potential of this new methodology of lineage analysis. Previous lineage analyses based on classical techniques (such as marking a cell and following its progeny) were unable to reconstruct lineage trees at the single cell level due to inherent limitations. This is now changed: in principle the lineage relations between any set of isolated cells can be reconstructed. The lineage trees shown here as well as the one reconstructed by Horwitz and colleagues [6] are just first examples, but we believe that a new field of cell lineage analysis will emerge. This possibility is also due to technological advancements: WGA of single cells provides large DNA quantities from practically any desired cell [28], and high-throughput DNA analysis technologies enable cheaper and quicker analysis of multiple loci obtained from WGA. In the long term we believe that this methodology and technology will drive large-scale projects such as the ‘‘Human Cell Lineage Project’’ (and its Mouse parallel [1]), aiming to understand ever larger portions of these organisms. We also foresee that this methodology will drive a conceptual change and create a new field of computational lineage analysis. The conceptual change is first of all based on the definition of a cell lineage tree (see [1] and this paper), which defines the lineage relations between cells according to their genealogical relations in this tree. The term lineage has acquired over time multiple meanings [29], which do not necessarily correspond to the rigorous definition of genealogical lineage. For example, it is used to define the acquisition of a state of commitment (e.g. ‘B- lymphocyte lineage [30]) or a specific tissue of origin (e.g. ‘mesodermal lineage’ [31]). Among developmental biologists, such distinctions are not always made explicit or even recognized [32]. Computational analyses of cell lineage trees coupled to biological or clinical understanding of these analyses are required to understand such trees. These analyses are necessary because lineage trees encapsulate a wealth of information, yet most of this information is not self-evident. Understanding the cell lineage trees of higher organisms, especially human, is a fundamental challenge of many branches of biology and medicine (see [1]). Several open questions are actually lineage questions in disguise, and knowledge of the relevant portions of the cell lineage tree could shed light on these questions. For example, in cancer research, lineage analysis can reveal the timing of tumor initiation, the relation between PLoS ONE | www.plosone.org 8 April 2008 | Volume 3 | Issue 4 | e1939