Surface Ectoderm Morphogenesis Figure 1. SE labelling induced from E6.5 to E13.5 reveals a single clonal strategy for all regions of the embryo. (A–C) [ROSAcre- ERT6R26R] and (D–F) [CT26R26R] embryos. Pregnant mice injected with 4-OHT at E13.5 (A), E12.5 (B), E9.5 (C), E8.5 (D), E7.5 (E), E6.5 (F). Observation of E14.5 embryos. Growth is isotropic (A); dorso-ventrally oriented and coherent in (B) to (D). In (E) and (F), growth is dispersive and results in longitudinally dorso-ventrally oriented stripes. (G), (I), (K–Q) Examples of clones observed in LaacZ embryos; (H), (J) Examples of clones observed in lox-LacZ embryos induced at E6.5. (G)–(J) in head regions, (K) to (P) in the trunk and (Q) in the tail. Arrowheads indicate the most dorsal position to which the clones contribute. doi:10.1371/journal.pone.0004353.g001 clones (Fig. 2L–L9), the labelling was mosaic in the SE, indicating that the SE is derived from several founder cells, and therefore that there are groups of clonally related P-DVCUs. The clones with the largest total cell number (Fig. 2A–D and L) made the largest contributions to the SE compared to clones with fewer cells (Fig. 2E–J). The size of the clone correlates with its date of birth and therefore the earliest ancestral cells produce more SE founder cells than the later ones. The labelling pattern in the large ancestral clones (Fig. 2A and C) included both sides and all regions of the SE; in the smaller ones (Fig. 2E–J) it was mostly restricted to only one side of the SE. The bilateral contribution of SE precursor cells is therefore restricted very early. The clonal pattern of the embryo in Fig. 2G is restricted to part of the DV axis of the SE. The contribution of SE founder cells is therefore DV restricted. This restriction occurs before the establishment of the pool of founder cells (compare Fig. 2E–F and G–H). Finally, all seven clones of ancestral cells exhibit an extensive AP labelling from the head to the tail. Therefore there is no obvious AP restriction of the ancestors and founder cells. The labelling shown in Fig. 2K–K9 is the most extensive restricted to the SE, found in the LaacZ library. The clone exhibits all the characteristics described for the labelling of the ancestral cells of the SE: it is unilateral, extends along the whole AP axis and its contribution to the DV axis is restricted. It may correspond to the labelling of a SE founder cell. To assess whether the whole SE can be produced from the founder cells descended from the seven ancestral clones, their contributions were superimposed on a single schematic represen- tation of an E14.5 embryo (Fig. 2M–M999). Labelling was observed in all AP and DV regions, including those formed late such as the posterior regions. The region above the neural tube was under represented (Fig. 2M, the region delimited by the lines). It is therefore not necessary to invoke recruitment from another source for any regions of the embryo including the late-formed structures; the pool of SE founder cells is probably a closed pool from an early stage of embryogenesis. These analyses showed that each cell of the initial pool of SE founder cells produces large groups of P-DVCUs. These P- DVCUs are not randomly distributed in the embryo; they are arranged longitudinally and their contribution is DV restricted. The most dorsal part of the SE is under-represented relative to all other regions. If it is produced from founders that are restricted along their DV axis, like the other founders, the corresponding pool is smaller. The ancestral cells of the SE are not equipotent. They can produce different numbers of organized founder cells whose properties are not equivalent. This reveals that the ancestral cells show a certain level of coherence and do not mix freely with the other cells of the embryo before their allocation to the SE. PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4353