Surface Ectoderm Morphogenesis Figure 7. Relationship between the pools of founder cells. (A)–(C9): examples of clones whose contribution is restricted to the head and the tail. (D)–(E9): examples of clones that contribute to both the trunk and posterior regions. (F): an example of a clone that stops at the level of the hindlimb. (A–A9, D, E–E9, F) from the E6.5 lox-LacZ library; (B–B9): spontaneous labelling; (C–C9) from the LaacZ library. doi:10.1371/journal.pone.0004353.g007 Discussion The combination of two complementary methods of cell labelling We combined two methods of cell labelling to provide a particularly powerful tool for describing cellular aspects of morphogenesis. The LaacZ system has been rendered ubiquitous by introducing the reporter gene into the ROSA26 locus [24]. The ROSA26- LaacZ method permits visualization of all cells of clones, including those outside the structure of interest; it also ensures that no territory is excluded from the analysis. Our study validates this approach, so that it can now be used to study more complex systems or intricate periods of development such as gastrulation. A system of production of labelling in which the birth date is controlled (GICL) [20] has also been made ubiquitous by using ROSA26-driven Cre recombinases. We used this system to generate polyclonal labelling and clonal labelling induced during gastrulation. The ability to generate polyclonal labelling allowed clonal saturation and therefore testing of the generality of the conclusions for any given structure. Polyclonal labelling can readily be generated using lines that present a moderate background of spontaneous recombination, as illustrated here with ROSA26cre-ERT [33]. The ability to generate clonal labelling allows saturating critical periods of development. Clonal labelling requires a Cre recombinase that presents a low frequency of spontaneous labelling such as the CT2 line. The CT2 line made possible the generation of a saturated E6.5 lox-LacZ library, in a way that would have been impossible with the LaacZ system. It also allowed classification of the LaacZ clones generated in the period before, during and after the period covered by the lox-LacZ library. By saturating at E6.5 we could validate two families of clones, those that participate only in the head and the posterior part of the embryo and those that derive from cells set aside early in development that contribute to the formation of the posterior part of the embryo only. This observation is critical to interpret SE morphogenesis. However, the general use (that is not restricted to early stages) of the temporal system of clonal labelling is limited by the spontaneous generation of labelling ([37,38]; this article). Nonetheless, the power of this approach is now demonstrated ([20]; this article), so it would be beneficial to find ways to abolish the spontaneous generation of labelling, to make it more generally applicable. Sophisticated modes of SE formation Morphogenesis of epithelial tissues generally involves an isotropic mode of cell proliferation [35,39]. This mode does not require either cell movement or cell orientation or other sophisticated cell behaviour [13]; it engages only minimal cellular operations and genetic control. Therefore, it would seem particularly adapted to the growth of the SE whose main known property is to cover the embryo. Indeed, it is this simple mode of growth that is observed in the SE after E14, even if the final size and shape have not been attained. However, before E14, the SE does not grow in an isotropic mode. Between E14 and E9, growth is in dorso-ventrally oriented stripes and from E6.5 to about E9 growth is dispersive, along lines parallel to the longitudinal axis of the embryo with clonally related cells interspaced. It is the completion of the spacing of the P-DVCUs that defines the transition between the period of growth of the DVCUS and the period characterized by these lines. The succession of two sophisticated modes of growth demonstrates that SE formation is PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4353