Surface Ectoderm Morphogenesis Figure 5. Cell arrangement of the clonally related P-DVCUs in the head. (A)–(G): Examples of clonal organisation in the head. The most dorsal positions of the DV-oriented stripes have been connected by one (A)–(C), two (D), (E) or three (F)–(G99) lines, defining four discrete DV positions: dorsal (A), dorso-lateral (B),(E), latero-medial (C) and mid-ventral (D), (F), (G). Points indicate the most dorsal position of the DVCU. The red lines connect DVCUS in the head, the magenta lines connect DVCUS in the trunk. (H), (I): Ventral labelling in the head. These labellings are connected with more dorsal head regions (arrowheads) but not with the ventral trunk region. (A), (F), (G) from the E6.5 lox-LacZ library; (B)–(E), (H)–(I): from the LaacZ library (F, F9, F99; G, G9, G99 and E, E9, E99 different views of the same embryo). doi:10.1371/journal.pone.0004353.g005 We examined clonal continuity between the head and the trunk. Among the 15 clones in the head, 9 extend to the trunk without change in the DV position of their DVCUs (Fig. 5A, B, D, F and G; magenta lines). The other six exhibit a shift at the neck level (Fig. 5C). Six of the 10 clones that have a short extension in the head also extend to the trunk, without change in their most DV position. Thus, 15 of the 25 clones show continuity of the most dorsal position of their DVCUs from the head to the trunk and therefore the regions defined in the trunk have a correspondence in the head. This applies to the dorso-lateral (Fig. 5B), the mid- ventral (Fig. 5D) and the dorsal lines (Fig. 5A, F G). However, the ventral region cannot be defined this way in the head: the ventral clones are genealogically closest to cells from more dorsal regions in the head (Fig. 5H–I, arrowheads). Note that this correspondence between the trunk and the head only concerns the step of AP dispersal of P-DVCU, not their contribution to the different DV regions. Although the trunk DVCUs are generally restricted to one (Figs. 5E, 3F) or two (Fig. 4E, E9) adjacent regions, this is not true for the head: most DVCUs contribute to several regions lateral to their most dorsal position, frequently as far as the most ventral head domain (Figs. 5H, 3J–L). This study reveals a general organization of the P-DVCUs in five sectors in the trunk and four positions in the head. These sectors that lie parallel to the main axis of the embryo, are respected during the AP dispersal of the P-DVCUs. These characteristics are observed whatever the mode of production of the P-DVCUs (sequential in the trunk and regional in the head). The dorso-ventral expansion of these longitudinal sectors forms the next DV regions in the SE. This DV expansion follows different rules in the head and trunk. In addition to this organization in longitudinal sectors, the clonally related DVCUs of a single sector are spatially separated along the AP axis; therefore we investigated the origin and uniformity of the spacing. Cell intercalation in the longitudinal sectors As the P-DVCUs are organized longitudinally, clonally related cells are expected to form continuous clonal columns in absence of cell rearrangement, irrespective of their mode of production. However this is not observed (Figs. 3–5). We studied the spacing of the DVCUs in clones in detail to assess the extent of the cell rearrangements. In all trunk DV regions and at all DV levels in the head, the clonally related DVCUs are well separated from one another (Fig. 6), evidence of cell rearrangement leading to cell intercalation of the P-DVCUs whether in the dorsal (Fig. 6A–E), dorso-lateral (Fig. 6B–F), latero-medial (Fig. 6C–G) and mid-ventral (Fig. 6D– H) regions for the head and trunk, and in the ventral (Fig. 6I) region for the trunk. The spacing is variable in all regions (Fig. 6. compare G to H) but the ventral region of the trunk usually exhibits moderate or no spacing (Fig. 6I, n = 4/5). Consequently, cells of different clonal origins intercalate to form a given sector. This intercalation takes place in a context of regional production in the head and of sequential production of the P-DVCUS in the trunk. Intercalation is advanced at E7.5 in the head, but is not finished and continues after this stage in the trunk. A more pronounced intercalation is observed in the dorsal and dorso-lateral than ventral regions of the trunk (Fig. 6. compare E, F to G, I), possibly indicating a lateral to medial direction of the cell rearrangement. Relationship between the head, trunk and tail pools of founder cells Finally, to determine the relationship between the pools of founder cells of the three AP regions, that is to understand how the trunk and tail pools of precursor cells are formed, we searched for clonal continuity between these three regions. Formation of the trunk pool. Eighteen of the 64 clones induced at E6.5 (lox-LacZ library) contribute to both the head and PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4353