elegans, in which there is a fixed relationship between cell ancestry and cell fate, lineage boundaries do not necessarily coincide with functional boundaries [3]. Two exceptions are its intestine and the germ line, both of which are identifiable tissues generated as ‘‘single exclusive clones’’ [3], i.e. in each case one precursor generates all the cells of the tissue and no other cell. Examples of partial clonal derivation are also present in C. elegans: some precursors generate only, but not all, cells of a given type; other precursors generate all, but not only, cells of a given type. However, throughout most of the lineage tree, cell types are intermingled even at the terminal divisions [3]. This is true for various invertebrate taxa, whose lineage trees show little correlation between the phenotypic similarity of two differentiated cells and the closeness of their genealogical relation [9]. While in C. elegans and other simple organisms the relation between lineage and function is completely known (though not necessarily understood), this relation is much less known in higher organisms. In order to address this, and other similar questions in mice, we defined a general scheme for lineage analysis which assigns statistical significance to the correlations between lineage and various cell properties (see Results). In addition to obtaining knowledge regarding the general features of the lineage tree, we also focused on a specific cell type–satellite cells–a small population of muscle stem cells residing beneath the basal lamina of each myofiber [10]. Satellite cells, which are normally quiescent, are activated in response to muscle injury, upon which they proliferate, differentiate and fuse together to repair or replace damaged myofibers [10]. Despite the extensive research, the exact lineage of satellite cells is unknown. We analyzed satellite cells in two respects: first, whether there is a correlation between the lineage of satellite cells and their physical proximity. Specifically, we ask whether cells which are physically closer share a significantly longer common developmental path. In general, sister cells can stay close to each other following cell division, a situation called coherent growth [11] which has been demonstrated in compartments [12] and in cancer [Frumkin D. et al., submitted]. Alternatively, sister cells can disperse as a result of cell motility, a common phenomenon in early embryogenesis of vertebrates [11], for example in the widespread dispersion of neurons across functional regions of the cerebral cortex [13]. Second, we checked whether there is a correlation between the lineage of satellite cells and anatomical proximity. The anatomy of skeletal muscle defines a hierarchy among its components: each muscle is made of many myofibers, on each of which reside several satellite cells. We defined anatomical proximity between satellite cells according to their relation within this hierarchy, and the hierarchy of the body (left vs. right lateralities). We analyzed whether satellite cells from the same myofiber, muscle or body-side have a significantly longer common developmental path than a random pair of cells. Results Cell Lineage Reconstruction We performed three lineage experiments on mice called ML2, ML4 and ML7 at the age of 10, 13 and 5.5 weeks respectively. In each experiment, we isolated single cells of multiple types (see complete lists in Tables S1, summarized in Table 1) and performed a semi-automated procedure (similar to that described in [1]) which yields lineage trees depicting their lineage relations (Figure 1C). In these experiments, genomic DNA from each cell was amplified (either by whole genome amplification or ex vivo culture) and then genotyped over a set of almost 100 MS loci (of various repeat units and lengths, see Table S2). Genotyping (performed automatically, see Text S1) assigns to each cell a vector, called an identifier [1], Cell Lineage Trees in Mice representing the mutations it accumulated at the set of loci in comparison to the zygote (see Tables S3a–c for complete cell identifiers; see Figure S1 for examples of MS mutations and signal analysis issues). Tail DNA was used to approximate the putative zygote of each mouse based on the assumption that the tail contains cells of multiple lineages whose most recent common ancestor (MRCA) is most probably the zygote, or one of its immediate descendants. Overall we identified 1156 mutations, accounting for 10.7% of analyzed signals. Cell identifiers served as input to the Neighbor-Joining (NJ) reconstruction algorithm [14] (applying the distance function ‘Absolute-distance’, see Materials and Methods). For analysis we chose informative samples that had at least 5% amplification and at least one mutation (ML7 oocytes were not included in the lineage analysis and will be reported separately with more comprehensive oocyte data). The output of this analysis for each experiment was a rooted binary tree depicting the inferred lineage relations between the set of sampled cells and the zygote, which includes intermediate nodes representing precursor cells of the sample (Figures 2–3). Beyond lineage relations, reconstructed trees also show the estimated depths of individual cells (the depth of a cell is the number of cell divisions it underwent from the zygote). These depths were obtained using a new method we recently developed, based on the correlation between genetic distance and cell depth, using an ex vivo cell cultured tree for calibration [15]. While reconstructed trees are enlightening in themselves, some analysis (see below) is required to understand the information they encapsulate. Lineage Analysis of Reconstructed Trees In this work we perform lineage analysis in an attempt to find correlations between the cell lineage tree and cell type and the anatomical proximity of satellite cells within the hierarchy of muscle. Our analysis tests whether cells within a certain set (e.g. B- cells or satellite cells from the same muscle) have on average a longer common developmental history than a random pair of cells (see Figure S2 for various theoretical outcomes in a lineage analysis checking the correlation between lineage and cell type). We use two measures between a pair of cells that are expected to correlate well with the length of the common developmental history of a pair of cells: the depth of the MRCA of the pair (as assigned by the reconstruction algorithm), and the percent of common mutations shared by the pair. The latter measure does not depend on a reconstructed tree, which has a speculative component in it, but rather on the ‘‘raw’’ data, i.e. the cell identifiers. We performed these analyses only in experiment ML7 because experiments ML2 and ML4 had a small sample of cells. Analyzed Cell Types in Mouse are not Single Exclusive Clones A quick glance at the reconstructed lineage trees of ML2, ML4 and ML7 (Figures 2–3) reveals that none of the analyzed cell types (as given in Table 1) is a single exclusive clone, since no cell type is completely and solely clustered on a subtree. We further investigated whether there may be a subtler correlation between lineage and cell type in ML7. We performed lineage analysis as described above (using both measures) and found that neither of the cell types (satellite cells and B-cells) is significantly correlated to length of common developmental history. Satellite Cells within a Myofiber Share a Longer Common Developmental History The mouse body contains a large number of muscles (on the left or right body side), each of which is made of a bundle of myofibers, PLoS ONE | www.plosone.org 3 April 2008 | Volume 3 | Issue 4 | e1939