Table 3. The lox-LacZ library of clones induced at E6.5 and observed at E14.5. 4-OHT dose Non injected 0,44 mg.g21 iv 0,33 mg.g21 iv 144 161 126 No of embryos No of embryos with with clones Total no of clones composed of composed of 200 embryos more than 400 cells to 400 cells 2% (3) 8% (13) 12% (15) x2 = 5,49 ; p = 0,019 x2 = 9,08 ; p = 0,0026 x2 = 10,4 ; p = 0,012 x2 = 22,7 ; p,0,0001 1% (2) 9% (15) 17% (21) The 64 SE clones in the library are from several series of injection. The x2 and Fisher’s exact probability tests (x2 corr) were used to compare the experimental groups with the control groups (first line). doi:10.1371/journal.pone.0004353.t003 the spontaneous activity of the inducible Cre recombinase must be low enough to allow the distinction between induced clonal labellings and background linked to spontaneous labellings. It appeared that the ROSA26cre-ERT line [33], that can be used to produce polyclonal labelling (see below), is improper for clonal labelling because of a significant level of spontaneous recombination. Another ROSA26 Cre line, CT2 (L. Grotewold and A. Smith, unpublished), in which a Cre-ERT2 gene has been introduced at the Nhe1 restriction site located 1 kb downstream of the usually used Xba1 restriction site, presents a frequency of spontaneous labelling compatible with the generation of a library of SE clones induced during gastrulation. Indeed the expected size of SE clones induced at E6.5 and observed at E14.5 is about 256 cells (28, assuming a doubling time of 24 h) and in [CT26R26R] embryos the frequency of spontaneous labelling of clones with 200 to 400 cells is only 1.361022 (Table 3, first line). This allows inducing clones at a frequency fifteen time above that of the background (up to about 261021). At this value, the frequency of double induction events is only 461022 (see Materials and Methods), that is too unfrequent to have an incidence on our analyses. A library of SE clones induced during gastrulation and referred to as the lox-LacZ library has been produced using 4-OHT concentrations yielding labelling frequencies between 1 and 261021 (Table 3). To further increase the stringency of the library, we look for potential variability in the induction frequencies by comparing litters 2 by 2 using Fisher’s exact test. The two extreme categories (the least labelled potentially not induced and the most labelled potentially too induced) were discarded. The statistical analysis of the final library (64 labelling among 287 embryos) that confirms its validity is reported in Table 3. Terminology In this article, the following terminology [3,9,23,34] is used. The ancestor cells of a structure are defined as any cell that will contribute at least some descendants to this structure and also to other structures in the embryo. The founder cells of a structure are defined as the first cells of a lineage whose contribution is restricted to that structure [34]. The extent of clone contribution to different structures and tissues of the embryo defines ancestral and founder clones. During coherent growth, sister cells remain close to each other, while dispersive growth results in widely separated sister cells. In addition growth can be either oriented or isotropic. The analysis of the spatial distribution of a clone informs about its mode of growth. In a library of clones, saturation is reached when the library has more than one example of any possible labelling pattern. At this stage, additional Surface Ectoderm Morphogenesis clones do not provide new information. The clonal complexity of a region is equal to the number of clones that contribute to this region. Clonal complexity can be used to detect mode of growth and territory of preferential growth in a structure [14]. For this kind of analysis, the structure of interest is divided in regions and contributions of the clones from a library at saturation to these regions are determined and compared. Cell behaviour during SE formation To determine the cell behaviour involved in the formation of SE, polyclonal labellings were first induced between E8.5 and E13.5 and observed at E14.5 ([R26CreERT xR26R] E6.5 to E13.5 libraries). After induction at E13.5, the labelling revealed coherent and isotropic groups of cells (Fig. 1A). Similar but more extensive labelling was observed in newborn mice. SE growth is therefore coherent and isotropic from E14 to post-natal or later stages, as expected [35]. After induction between E8.5 and E12.5, coherent stripes of cells oriented dorso-ventrally were labelled, and were larger when induction was earlier (Fig. 1B–D). Thus, SE growth between E9 and E14 is coherent and oriented. Then clonal labellings were induced between E6.5 and E7.5 and observed at E14.5 ([CT26R26R] E6.5 and E7.5 libraries, hereafter called the lox-LacZ libraries). Clonal induction between E6.5 and E7.5 resulted in groups of a few DV-oriented stripes distributed along the AP axis (Fig. 1E induced at E7.5 and F induced at E6.5). A period of cell dispersion along the longitudinal axis of the embryo therefore precedes the period of coherent and oriented growth along the DV axis. This period corresponds to the first stages of elongation of the embryo. These findings suggest a clonal strategy involving a mechanism that distributes cells longitudinally, then a mechanism that arrests cell dispersion and produces oriented stripes, followed by a mechanism that shifts oriented growth to isotropic growth. The clonal signature of this strategy is the DV oriented stripes. The same clonal strategy is used in all regions of the embryo We next investigated whether this clonal strategy is used in all regions of the SE. Therefore we searched for clones composed of a single DV stripe in the LaacZ and lox-LacZ libraries. Such clones were found in all regions of the head: the facial region (Fig. 1G), the encephalic region (Fig. 1H), the maxillary region (Fig. 1I) and the neck (Fig. 1J); the trunk, dorsal (Fig. 1K–M), lateral (Fig. 1N, O) or ventral (Fig. 1P), from the anterior limit of the forelimb (Fig. 1K) to the posterior limit of the hindlimb (Fig. 1O); and the tail (Fig. 1Q). The presence of this clonal signature in all regions of the embryo suggests that the same cell behaviour is involved in all SE regions. We named the precursor cells of the DV-oriented stripes Precursor of DV-oriented Clonal Unit (P-DVCU) and the DV- oriented clonal unit DVCU. We next followed the clonal history of these precursors to determine how they are produced and positioned longitudinally and dorso-ventrally and whether they are governed by different modes of cell behaviour. A pool of SE founder cells already regionalized for its dorso-ventral contribution We used the LaacZ library of E14.5 embryos to study how the pool of SE founder cells (see terminology) is formed. As founder cells derived from ancestral cells (see terminology), we first searched for ancestral clones. Seven clones contributing to both SE and internal structures (Fig. 2A–J9 and L–L9) that is, labelled before the restriction of cells to the SE, were found. In six PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4353