Surface Ectoderm Morphogenesis Figure 6. Spacing of the clonally related P-DVCUs. (A)–(D): Examples of spacing in the head for the four DV positions: (A), dorsal, (B), dorso- lateral; (C), latero-medial and (D) medial. In D the magenta point indicates a second dorsal line of dispersion. (E)–(I): Examples of spacing in the trunk for the five DV positions dorsal (E), dorso-lateral (F); latero-medial (G, red points); medial (G, magenta points and H); and ventral (I). Note that the spacing is greater dorsally than ventrally. (A), (E), (G), (I) from the E6.5 lox-LacZ library; (B)–(D), (F): from the LaacZ library; B and F are two views of the same embryo; (H): spontaneous labelling. The points represent the most dorsal position of the DVCUs. doi:10.1371/journal.pone.0004353.g006 the trunk (region 1–5 and 6–9 respectively, Fig. 3B, C–E; Fig. 5A, F, G). Therefore there are still common precursors at E6.5–E7.5 for both structures on which the head-trunk clonal continuity is built. In ancestral clones from the LaacZ library, no clones that contribute to the trunk but not to the head were found (n = 0/7). Therefore, all clones that contribute to the trunk also contribute to the head (Fig. 2A–G, K), indicating that perhaps all of the trunk pool cells are derived from precursor cells common to the head and the trunk. The library of 53 clones (with estimated date birth between E6.5 and E7.5) was searched for clones contributing to the head but not to the trunk, to determine if all founder cells of the head SE contribute to the trunk pool. Six such clones were found: they participate in the head and the tail and/or the posterior part of the trunk, but not to the region between the forelimb and the hindlimb (Fig. 7A–C9). These clones reveal the existence of head SE founder cells that do not contribute to the trunk pool. The clones, which show several lines of dispersion in the head, provide additional information. Among the bilateral clones in the head, one line of dispersal always ends in the region between the external ear and the forelimb (n = 11/11, Fig. 5E9). In the most complex labelling in the head SE, with three lines of dispersal (n = 3/9), one (Fig. 5F9) or even both (Fig. 5G, G9) lines on the sides stop, again in the region between the ear and the forelimb. This shows that precursor cells, common to the head and the trunk, produce descendant cells that contribute only to the head (represented by clones with the lines that do not extend to the trunk). Most lines (n = 17/28) that extend from the head to the trunk are dorsal or dorso-lateral (Fig. 5A, B, E, F, G). Moreover, the lines that extend from the head to the trunk appear frequently to shift their position at the level of the neck. The shift is in all cases directed ventrally (n = 17/17, Fig. 5C), sometimes with a shift of more than one adjacent position, cells shifting from dorso-lateral to mid-ventral, for example (Fig. 5E). Therefore the trunk pool is formed of cells that have common ancestors with the cells that constitute the head SE, including its most anterior part. These precursor cells form a common head- trunk pool at E6.5–E7.5 that constitutes the main and possibly only clonal origin of the trunk pool. It also generates precursor cells that contribute only to the head SE. It is mainly the cells that have a dorsal or dorso-lateral position in the head that form the trunk pool. The lateral regions in the trunk are in part formed by ventrally shifted cells that are more dorsal in the head. Formation of the posterior pool. The most posterior part of the trunk is produced by a restricted pool of cells set aside between E6.5 and E7.5 (lox-LacZ library: Fig. 3A, clones 61 to 64, Fig. 3I). Six non-restricted clones found in the lox-LacZ and the LaacZ libraries exhibit labelling only in the head and the tail (Fig. 7A–C9). It suggests that at least part of the posterior pool is derived from the regionalization of founder cells (represented by these six clones) whose participation is restricted to the head and the tail. The trunk pool distributes clones up to the posterior limit of the hindlimb, or beyond (Fig. 3A). We searched the library of 53 clones for examples contributing to both the trunk and these posterior regions, and four such clones were found. Two contribute only to the proximal part of the tail (Fig. 7D) and the other two, to both the proximal and distal parts of the tail (Fig. 7E– E9). The other four clones in the library that participate in the same region of the trunk all stop at the level of the hindlimb (Fig. 7F). Thus, the pool of cells that participate in the anterior trunk can also contribute to the posterior trunk (4 out of 9), but rarely to the most distal part of the tail. Very similar clones were also found in the LaacZ library. In conclusion, the posterior pool is formed partly by cells from the trunk pool and partly by a pool set aside at E6.5–E7.5 of which at least a part is derived from the regionalization of SE founder cells restricted to the head and the tail. PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4353