Surface Ectoderm Morphogenesis posterior region to which they contribute for two representative models for their production: self-renewing pool of cells (P); and from the regional mode (P9). Each column (of different colour) represents a clone. (Q)–(R) Schematic representation, of the LaacZ clones that contribute (Q) to regions 6 to 9 (see B); (R) to regions 1 to 5 (see B). Clones were classified according to size (long on left and short on right) and the most posterior region to which they contribute. Note that clones from ancestral cells of the SE (shown in O) have been removed. (Q) Long clones that contribute to all regions are present on the left. (R) Clones from ancestral cells of head SE are shown on the left. Note the absence of clones that contribute to all five sub- regions. doi:10.1371/journal.pone.0004353.g003 Figure 4. Cell arrangement of the clonally related P-DVCUs in the trunk. (A)–(G): Examples of clonal organisation in the trunk. The most dorsal positions of the DV-oriented stripes have been connected by one (A)–(D), two (E) or several (F), (G) lines, defining five discrete DV positions: dorsal (D), dorso-lateral (A), latero-medial (A, magenta line), mid-ventral (B), and ventral (C), schematically represented by yellow lines in (H and H9). A– A9, B and E–E9, from the LaacZ library; C, D, from the E6.5 lox- LacZ library; F, G: spontaneous labelling. A–A9 and E–E9 are two different views of the same embryo. d: dorsal, dl: dorsal-lateral, lm: lateral-medial, mv: mid-ventral, v: ventral. doi:10.1371/journal.pone.0004353.g004 tendency to shift from ventral to medial during AP dispersion (see below). In these last two cases, the shifts do not seem random and may correspond to defined DV regions of the embryo. Thus, the labelling in the trunk reveals a clonal pattern, organised parallel to the AP axis of the embryo. The P-DVCUs produced by a single cell in the posterior pool contributes to the same longitudinal line. Shifts are observed, but only in a small number of clones and can be attributed to the behaviour of the cells in the posterior pool. The shifts (Fig. 4E–G) suggest longitudinal organization and also organization in a defined dorso-ventral sector. We analyzed the DV positions adopted during AP dispersal of the 56 long lines of the clones with one or two longitudinal lines. The lines are found in only five DV positions (Fig. 4H, H9). Twenty-four lines are in a dorsal position, lateral to the dorsal midline of the embryo (Fig. 4A). This position dorsally limits a dorso-lateral region. Eleven lines are in a more lateral position that delimits a latero- medial region (Fig. 4E, magenta line). Nine lines characterize a more lateral position, the stripes being ventral to the limbs, limiting dorsally a mid-ventral region (Fig. 4B). Five lines characterize a fourth position (Fig. 4C), referred to as ventral, that limits dorsally a region that reaches the ventral midline. Seven lines characterize the last position: the middle of their DV stripes approximately coincides with the midline of the embryo (Fig. 4D). This position is referred to as dorsal. Therefore the trunk is characterized by only five longitudinal sectors. For the clones exhibiting two lines or shifts, the lines were always in adjacent positions, for instance dorso-lateral and latero- medial (Fig. 4E), or mid-ventral, latero-median and dorso-lateral (Fig. 4G). This applies to almost all ‘‘shifted’’ clones (n = 21/23; 41/44 shifts). In the head (regions 1 to 5, Fig. 3B), among the 15 clones (of the 25 that contribute to the head in the library of 53 clones) that have an extension sufficiently long to define the line that connects the dorsal limit of their DVCUs, 12 have only one line. The line is, as in the trunk, parallel to the longitudinal axis of the embryo (n = 12/15, Fig. 5A–C red lines). Two of the other three clones show two lines parallel to the AP axis and the last one shows a shift (Fig. 5D red line). The AP dispersion therefore respects the relative DV position of the clonally related DVCU in all cases except one, and, as in the trunk, the organization is longitudinal and parallel to the embryonic AP axis. Nine of these 15 clones show bilateral labelling in the most rostral part of the head (Fig. 5E–E’’). In most cases (n = 5/9), the lines are at the same DV level on either side of the embryo, dorso- lateral (Fig. 5E–E9) or latero-ventral. Similar labelling was observed in clones that contribute only to the head. This suggests that the dispersion of the cells at the origin of the left and right contribution follows an identical line and continues unchanged rostrally (Fig. 5E–E9). The most complex clones in the head can be resolved by adding one further line that generally links very dorsal DVCUs (Fig. 5F–G99, n = 3). PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4353