Large-Scale Clonal Analysis Reveals Unexpected Complexity in Surface Ectoderm Morphogenesis Anne-Ce´ cile Petit, Jean-Franc¸ ois Nicolas* Unite´ de Biologie mole´culaire du De´veloppement, Institut Pasteur, Paris, France Abstract Background: Understanding the series of morphogenetic processes that underlie the making of embryo structures is a highly topical issue in developmental biology, essential for interpreting the massive molecular data currently available. In mouse embryo, long-term in vivo analysis of cell behaviours and movements is difficult because of the development in utero and the impossibility of long-term culture. Methodology/Principal Findings: We improved and combined two genetic methods of clonal analysis that together make practicable large-scale production of labelled clones. Using these methods we performed a clonal analysis of surface ectoderm (SE), a poorly understood structure, for a period that includes gastrulation and the establishment of the body plan. We show that SE formation starts with the definition at early gastrulation of a pool of founder cells that is already dorso-ventrally organized. This pool is then regionalized antero-posteriorly into three pools giving rise to head, trunk and tail. Each pool uses its own combination of cell rearrangements and mode of proliferation for elongation, despite a common clonal strategy that consists in disposing along the antero-posterior axis precursors of dorso-ventrally-oriented stripes of cells. Conclusions/Significance: We propose that these series of morphogenetic processes are organized temporally and spatially in a posterior zone of the embryo crucial for elongation. The variety of cell behaviours used by SE precursor cells indicates that these precursors are not equivalent, regardless of a common clonal origin and a common clonal strategy. Another major result is the finding that there are founder cells that contribute only to the head and tail. This surprising observation together with others can be integrated with ideas about the origin of axial tissues in bilaterians. Citation: Petit A-C, Nicolas J-F (2009) Large-Scale Clonal Analysis Reveals Unexpected Complexity in Surface Ectoderm Morphogenesis. PLoS ONE 4(2): e4353. doi:10.1371/journal.pone.0004353 Editor: Mai Har Sham, The University of Hong Kong, China Received August 12, 2008; Accepted December 26, 2008; Published February 6, 2009 Copyright: 2009 Petit 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 work was supported by grants from Cells into Organs (European Network of excellence), EuroStemCell (European consortium for stem cell research) and the Association Franc¸aise contre les Myopathies. J.F.N. is from the Institut National de la Recherche Me´dicale, and A.C.P. from Universite´ Pierre et Marie Curie. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jfnicola@pasteur.fr Introduction In complex development, cells of the embryo are rearranged by cell movement and other cell behaviours [1,2] that shape the embryo and generate structures. Amniotes development occurs during periods of intense cell proliferation. As a result the signals to which cells are exposed change, with two consequences. It increases considerably the repertoire of combinatorial signals that the embryo can exploit, an evolutionarily favourable outcome. It generates the need for tight control of cell rearrangement and changes in shape, imposing major constraints on developmental processes [3]. How cell behaviour is exploited for morphogenesis and coupled to cell specification are major issues in developmental biology and are also of importance for the understanding of cellular operations evolution and their genetic control in animal groups [4,5,6]. Analysis of the contribution of cell rearrangement and movement in mouse morphogenesis by following the embryo in vivo is difficult because of its inaccessibility and the impossibility of long-term culture [7,8]. Genetic methods of clonal analysis [9,10,11,12] present an alternative that can provide information about cell proliferation, mode of growth, cell rearrangement and other aspects of cell behaviour [13,14,15,16,17,18,19,20,21]. We improved two clonal analysis methods: the LaacZ method of random induction of labelling [11,14,19,22,23] that has been modified to make all cell types visualizable [24]; a method of genetic induced cell labelling [20], abbreviated GICL in this article, adapted from genetic induced fate mapping (GIFM) techniques [25], that allows temporal induction of the labelling and has been modified to permit the labelling of any cell in the early embryo, all cell types being also visualizable. Combined together, these two methods permit large-scale production of labelling. We present an analysis, using these methods, of the formation of surface ectoderm (SE) from E6.5 to E14.5, a period that includes gastrulation and the establishment of most structures of the organism. We report that SE, a simple 2D monolayer epithelial structure, shows non-random cell behaviours, namely that SE formation and elongation involve different combinations of cell rearrangement and modes of cell proliferation according to position along the axis. Our results suggest that the posterior zone in the embryo is crucial for SE elongation; cell proliferation and cell rearrangement are temporally and spatially organized in this zone. Another finding is that there is an early common pool of precursors restricted to the head and the posterior part of the PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4353