Reconstruction of Cell Lineage Trees in Mice Adam Wasserstrom1, Rivka Adar1, Gabi Shefer5, Dan Frumkin1, Shalev Itzkovitz2, Tomer Stern2, Irena Shur6, Lior Zangi3, Shai Kaplan1, Alon Harmelin4, Yair Reisner3, Dafna Benayahu5, Eldad Tzahor3, Eran Segal2, Ehud Shapiro1,2* 1 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2 Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel, 3 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel, 4 Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel, 5 Department of Cell and Developmental Biology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel Abstract The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance. Citation: Wasserstrom A, Adar R, Shefer G, Frumkin D, Itzkovitz S, et al. (2008) Reconstruction of Cell Lineage Trees in Mice. PLoS ONE 3(4): e1939. doi:10.1371/ journal.pone.0001939 Editor: Thomas Zwaka, Baylor College of Medicine, United States of America Received November 7, 2007; Accepted February 25, 2008; Published April 9, 2008 Copyright: 2008 Wasserstrom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by The Israel Academy of Science and Humanities (Bikura), by The Yeshaya Horowitz association through the center for Complexity Science, by The Research Grant from Dr. Mordecai Roshwald, by The Grant from Kenneth and Sally Leafman Appelbaum Discovery Fund, by The Estate of Karl Felix Jakubskind, by The Clore Center for Biological Physics, and by a Research Grant from The Louis Chor Memorial Trust. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Ehud.Shapiro@weizmann.ac.il Introduction All the cells in the body of a multi-cellular organism, such as a human or a mouse, descend from a single cell–the fertilized egg. The exact history of cell divisions that an organism underwent since its beginning is naturally represented by a mathematical tree, which we call the organism cell lineage tree ([1], Figure 1A). Lineage trees encapsulate a wealth of information regarding the development and maintenance of the various subsystems of these organisms under physiological and pathological conditions. Lineage analyses, aiming to elucidate various parts of lineage trees, have been performed until now using a variety of methods. Direct observation of cell divisions enabled the complete reconstruction of the lineage tree of somatic cells of Caenorhabditis elegans [2,3], yet this method is inapplicable to humans and mice since they are opaque and have a tremendous number of cells [4]. A variety of methods for lineage analysis, generally termed clonal assays (reviewed in [5]), rely on marking some cells and tracing their progeny. These methods have yielded many insights, but can provide only course-grain information about a cell lineage tree [1,4]. In addition, these methods are also inapplicable to the study of humans because they are invasive. A new approach for cell lineage analysis was recently proposed by our group [1] and later independently by another research group [4,6]. This approach exploits stochastic processes that occur during the normal development of higher organisms. Before a cell divides, its genome is duplicated with very high precision, yet several mistakes, known as somatic mutations, occur in this process. These mutations are random and sufficiently rare such that they usually do not disrupt the functionality of the cell. Nevertheless, they contain very valuable information, as cells that share a common developmental path tend to share the mutations that occurred along this path. Our analysis shows that in higher organisms such as human and mouse the information available in such somatic mutations is rich enough to implicitly encode the entire cell lineage tree of an organism with very high precision ([1], Figure 1B). The fact that species that share a long evolutionary path tend to have similar genomes compared to species that have diverged earlier in evolution enables phylogenetic analysis at the species level. This similar phenomenon in cells of a multicellular organism enables the application of phylogenetic analysis to reconstruct the lineage relations between cells [1]. Because somatic mutations are relatively rare events, our analysis focuses on microsatellites (MS; repetitive DNA sequences with relatively high mutation rates) in mismatch repair (MMR)- deficient organisms. Such organisms have a much higher rate of MS mutations [7,8] without compromising their normal develop- ment. Another application of this approach was also demonstrated PLoS ONE | www.plosone.org 1 April 2008 | Volume 3 | Issue 4 | e1939