epithelium must be maintained, until the transition to the isotropic mode of growth. Head-tail clones, vestiges of the radial to bilateral transformation in bilaterians? The single clonal origin of the precursor cells for the trunk SE contrasts with the double clonal origin of the precursor cells for the head SE and for the SE of the posterior part of the embryo. Indeed, some head founder cells and some tail founder cells do not give rise to, or are not derived, from trunk founder cells: they are head and/or tail specific. Surprisingly, these two specific pools have (at least in part) the same clonal origin as evidenced by clones that contribute only to the head and the tail. A pool of common cells for anterior and posterior SE is therefore present early. This pool may contribute to as much as half of the founders of head and tail SE. Considering the origin of the axial tissues in bilaterians [62,63,64] perhaps helps explain this observation. In the common ancestor of deuterostomes and maybe even in the common ancestor of both deuterostomes and protostomes [65], the closure of the blastopore involves movement of lateral cells that separate the future anterior region (where the stomodeum will form) from the future posterior regions (where the anus will form). This cell movement closing the blastopore facilitates the radial to bilateral transformation of the embryos [66] and the appearance of the dorsal neural plate in deuterostomes and of the ventral neural cord in protostomes. We propose that this movement has been conserved in vertebrates and that it may have facilitated a new mode of cell proliferation (the sequential mode) for trunk elongation in an intense cell proliferation context. The clones restricted to the head and tail SE, the existence of three pools of cells at the origin of the longitudinal organization of the SE and the mediolateral convergent extension of the SE in the mouse may be the consequence of this founder episode. Materials and Methods Transgenic mouse lines The R26LaacZ1.1 line (from Elena Tzouanacou and Valerie Wilson) was obtained by introducing a LaacZ reporter gene by homologous recombination (HR) into the ROSA26 locus; the size of the duplication is 1117 bps. The CT2 line (from Lars Grotewold and Austin Smith) was obtained by introducing the CreERT2 gene [31] by HR into the ROSA26 locus. The ROSA26cre-ERT line was from Anton Berns [33] and the R26R Cre reporter mouse from Philippe Soriano [26]. In this line, a loxP-flanked-stop- sequence (PGKneo-polyA) upstream from the LacZ gene was introduced into the ROSA26 locus by HR. The ROSA26 promoter confers ubiquitous expression on LacZ, CreERT or CreERT2. Generation and observation of embryos [R26R6ROSA26CreERT2] and [R26R6ROSA26CreERT] embryos were obtained by crosses between CT2 or ROSA26cre- ERT males and superovulated R26R females. LaacZ embryos were obtained by crosses between homozygous R26LaacZ1.1 males and C57Bl/6 or Swiss superovulated females. Embryos were staged with the day after crossing being defined as embryonic day of development (E) 0.5. Embryos were dissected in PBS, fixed by incubation in 4% paraformaldehyde for 30 minutes, rinsed twice in PBS, stained in X-gal solution (4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6, 2 mM MgCl2, 0.5 mg.mL21 X-Gal in PBS) at 37uC for 48 hours to reveal b-galactosidase activity, rinsed twice in PBS and postfixed by incubation in 1% paraformaldehyde. Surface Ectoderm Morphogenesis 4-OHT preparation and injection Initially, 4-OHT was prepared in a hydrophobic solvent and injected intraperitoneally (ip) as in [67]. 4-OHT was suspended at a concentration of 100 mg.mL21 in 100% ethanol, diluted in autoclaved corn oil to 10 mg.mL21, sonicated for 30 minutes and stored at 220uC. Before injection, the suspension was diluted in corn oil to the desired concentration and vortexed. This protocol was used to prepare 4-OHT for polyclonal labelling in [R26R6ROSA26- CreER T ] embryos using 66 mg.g21 (ip). Subsequently, we used a novel protocol [36] involving cremophorH EL (Sigma) and intravenous (iv) injection. 4-OHT was diluted to 20 mg.mL21 in 100% ethanol, then diluted in cremophorH EL (Sigma) to 10 mg.mL21, and again in 1X PBS to 1 mg.mL21. Before injection, the suspension was diluted in 1X PBS to the desired concentration. Labelling was initiated by the intravenous injection (into the vein of the tail) of 4-OHT into pregnant mice at various times after coitus. This protocol was used to prepare 4-OHT for clonal labeling at E6.5 in [R26R6CT2] embryos using 0,44 and 0,33 mg.g21 (iv) (Table 3). Statistical analysis of the LaacZ library of clones The labeling of a cell by spontaneous intragenic homologous recombination within the LaacZ gene is a random event [11]. The frequency of N independent recombination events can be calculated by the fluctuation test of Luria-Delbru¨ ck [68]. The expected number of embryos with N independent recombination events is N0 (ln (Ne/N0))N/(N!), where N0 is the number of embryos observed with no recombination event (N = 0) and Ne the total number of embryos of the sample. Table 1 gives the calculated number of embryos with N events of recombination for a sample of 97 embryos of the LaacZ library and the observed number of embryos with possibly N recombination events. The frequency of events corresponding to the combination of two labelings of two different categories, A and B, equals the product of the probability of each single event. That is 2 C = NA6NB/(Ne) , where is the number of observations of event A, NB of event B, and Ne the total number of embryos in the library. Table 2 gives the number of events for each size category for the sample of 97 embryos and for the 4248 embryos of the entire library. Statistical analysis of the lox-LacZ library of clones induced at E6.5 The clonality of a labeling is documented by statistical tests. For a SE library of E6.5 lox-LacZ clones observed at E14.5 the 8 expected size of the clones induced is 256 cells (that is, 2 , assuming a doubling time of 24 h) or more (assuming a doubling time shorter than 24 h). In preliminary experiments we tested and confirmed the hypothesis that the number of clones in these categories (and not the others) is effectively increased. The E6.5 lox-LacZ library has been produced using concentrations of 4- OHT (0.33 to 0.44 mg.g21) that give a frequency of labelled embryos between 1 and 261021 (Table 3). In this condition, the probability of two independent events is 461022 (according to the formula C = NA6NB/(Ne)2, where = NB = 261021, see above) which is practically negligible and possibly identifiable (the second event being potentially anywhere in the embryo including in the contralateral side to the first labeling). To validate the library, spontaneous labeling of the category of clones to be induced must be substantially lower than 261021. The frequency of spontaneous labeling of clones with 200 to 400 cells was actually only 1.361022 (2/144) and that of clones with more than 400 cells was 261022 (3/144) (Table 3, first line, non- injected controls). PLoS ONE | www.plosone.org 13 February 2009 | Volume 4 | Issue 2 | e4353