The initial pool of SE founder cells is regionalized into three between E6.5 and E7.5 To examine how the pool of founder cells produces the SE, we generated a lox-LacZ library of clones induced at E6.5 in [CT26R26R] F1 embryos. Each of the 64 clones (Fig. 3A) had more than 200 cells. We estimate that the clones are born between E6.5 and E7.5, taking into account the delay and the time of action of 4-OHT [36] and the asynchrony of the embryos within and between the litters. Three clones that contribute to structures from the head to the base of the tail (Fig. 3C–D) are similar to the LaacZ clones that correspond to the labelling of founder cells (Fig. 2K–K9). The SE founder cells are therefore still present in the embryo at E6.5. The other clones contribute to the head and the trunk (Fig. 3E) or are restricted to a part of the AP axis: from the forelimb to the hindlimb (Fig. 3F), the posterior part of the trunk (Fig. 3G–I) or the head (Fig. 3J–L). These clones have no equivalent among the clones from the ancestral and founder cells of the SE; they have all the characteristics of sub-clones of the clones of founder cells, as each can be related to at least one of them (Fig. 3, compare E–H with Fig. 2K; G, J, K and L with C; I and J9 with D). This suggests that the initial pool of founder cells is becoming regionalized along the AP axis, defining a pool for the head (regions 1–5 Fig. 3B, clones 28 to 53, Fig. 3A), a pool for the trunk (regions 6–9 Fig. 3B, clone 20 to 27 and 54 to 56, Fig. 3A) and a pool restricted to the posterior regions (regions 9 and 10 Fig. 3B, clone 57 to 64, Fig. 3A). This posterior pool is unexpected as it is set aside at E6.5–E7.5 prior to the formation of the posterior regions of the embryo from E9. The smallest lox-LacZ clones are composed of single DVCU in the head (Fig. 3L) but of groups of seven to 20 DVCUs in the trunk (Fig. 3F) and the tail (Fig. 3I). Therefore, some direct precursors of head DVCUs are present in E6.5–E7.5 embryos although only precursor cells of groups of trunk and tail DVCUs are present. These results indicate: 1) The SE founder cells are present in E6.5 embryos. 2) This initial pool is rapidly regionalized into anterior, truncal and posterior regions. 3) In the anterior region, some of the direct precursors of DVCUs have already been produced although in the more posterior regions only precursors of large groups of DVCUS have been produced. This suggests that the production and individualization of the P-DVCUs progresses in a rostral to caudal direction. Head and trunk P-DVCUs are produced by regional and sequential modes respectively P-DVCUs may be produced in the regions defined between E6.5–E7.5 in a regional mode or a sequential (self-renewing) mode. As the process of production proceeds in a rostral to caudal direction (see above), the modelling of the sequential mode of clonal growth for clones generated by random events (the LaacZ library) has been based on the functioning of a posterior pool. In a sequential mode, in which a posterior pool of precursor cells produces P-DVCUs during the establishment of the AP axis, long clones would be expected all contribute to the posterior pole of the embryo and clonal complexity (see terminology) would be expected to increase from anterior to posterior. Only small clones distributed homogenously along the axis of the embryo, and no intermediate-sized clones, would be expected (Fig. 3P). In a regional mode, the SE would form from the expansion of a few regions defined early. No clones contributing to the whole axis would be expected (the only long clones would be those derived from the ancestor cells) and clones would be intermediate-sized and smaller, and distributed homogenously along the AP axis. There would be no regions with greater clonal complexity than Surface Ectoderm Morphogenesis others (Fig. 3P9, in which the SE would be formed from two regions). The distributions and the sizes of the clones of the LaacZ library (Fig. 30) were assessed and classified according to their most- posterior limit (Fig. 3Q, R) which corresponds to the most stringent condition for discriminating between the two models. The distribution of the clones in the region from the anterior part of the forelimb to the anterior part of the hindlimb (regions 6 to 9, Fig. 3B) was consistent with a sequential mode of growth (Fig. 3Q), this includes numerous long clones (Fig. 3Q, the 13 clones on the left) that contribute to the most posterior regions (regions 8 and 9) and many small clones distributed homogenously in the region. No intermediate-sized clone was observed. The distribution of clones in the head and neck (regions 1 to 5, Fig. 3B), however, was consistent with a regional mode (Fig. 3R): all clones were of intermediate size or smaller and were distributed homogenously in the region. All clones that contribute to all head-neck region result from labelling of the ancestor cells of this region (Fig. 3R, clones on the left). These results suggest that two different modes of growth are used for the SE during AP elongation: a regional mode for the head-neck region and a sequential mode for the trunk, from a posterior pool. The boundaries between the regions, however, are not sharp. Some clones that contribute to the neck also contribute to the sequential production of the trunk (Fig. 3Q, clones at left) and clones that contribute to the trunk can also contribute to the tail (Fig. 3Q, 3 clones at left); the intermediate regions may be produced by a mixture of both modes of growth. These observations do not describe completely the relationship between the P-DVCUs. This relationship is also dependent on dispersal properties of the cells in the regions considered. Precursors of DVCUs are dispersed along discrete longitudinal parasagittal lines along the AP axis In order to analyse the dispersion of clonally related P-DVCUs during elongation, we determined the positions of the DVCUs of clones. A line was drawn, connecting the dorsal positions of the DVCUs of clones that have estimated birth dates between E6.5 and E7.5. We studied the 14 long LaacZ clones, the 27 most extensive clones in the E6.5 lox-LacZ library and 12 spontaneous clones in [CT26R26R] embryos, all containing more than 400 cells. These clones will be referred to as the library of 53 clones. For 27 of the 50 clones labelled in the trunk (regions 6 to 9, Fig. 3B), a single line traced parallel to the longitudinal axis of the embryo connected almost all DVCUs from the most anterior to the most posterior (n = 27/50, Fig. 4A–D). The DVCUs have therefore been produced by the same single cell in the posterior pool (defined in the paragraph above): this cell would have kept a constant DV position during the period of AP dispersion. For another 14 of these 50 clones, two lines traced parallel to the longitudinal axis of the embryo, were required to account for the observed labelling: a major line that starts at the most anterior position and a secondary line that starts more posteriorly (Fig. 4E, E9 red and magenta lines). It is likely that the cell at the origin of the secondary line was produced in the posterior pool by the cell at the origin of the major line (such that they are clonally related) and has shifted to a more ventral position in the posterior pool. Then the two cells in the posterior pool have kept a constant position during the production of the SE. For another four of these 50 clones (Fig. 4F), the labelling shows groups of DV stripes spaced along the AP axis and shifted along the DV axis. The cell in the posterior pool probably moved during the production of the P-DVCUs. For the remaining 5 of the 50 clones, the labelling exhibits successive and ordered shifts along the DV axis (Fig. 4G); the cells of the clones show a strong PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4353