Surface Ectoderm Morphogenesis Table 1. The LaacZ library: frequency of embryos with N events of recombination. No of embryos with N recombination events expected No of embryos with N clones observed embryo. This puzzling observation is consistent with ideas about the origin of the axial tissues in bilaterians. Results The global LaacZ method and the SE LaacZ library The LaacZ method has been made ubiquitous by introducing a LaacZ reporter gene into the ROSA26 locus [24]. The ROSA26 promoter confers ubiquitous expression of LacZ [26]. The 1117bp duplication in the coding sequence of the LaaZ gene generates multiple in-frame stop codons. As a consequence, the LaacZ gene encodes a non-functional b-galactosidase and non-sense mediated decay is induced [27]. A functional LacZ gene can be restored by spontaneous intragenic homologous recombination within the duplicated region. The recombined LacZ is then transmitted to all descendants of the modified cell. The resulting clone is detectable by b-galactosidase histochemical staining [22,28]. The ROSA26- LaacZ method allows visualization of any clonally-related cell [24]; it therefore ensures that no area of the structure of interest is excluded from the analysis. A SE LaacZ library has been produced. It contains 4248 E14.5 embryos. To validate the library, a sample of 97 embryos has been screened for determining the number of embryos lacking SE labelling. 42 (44%) such negative embryos were found. From this number, the expected number of labellings corresponding to N recombination events (from 1 to 4) was calculated using the fluctuation test of Luria and Delbru¨ ck (see Materials and Methods). 35 (36%) embryos are expected to show clonal labelling (N = 1); 15 (15%), two recombination events (N = 2) and only 4 (4%), three recombination events (Table 1, left column). Owing to the independent nature of the recombination events in both time and space, most of the double or triple recombination events correspond to situations readily recognizable by the size and spatial disposition of labelled clusters. Indeed most double events involve a small second clone and most non-clonally related clusters are scattered in SE. In addition these composite patterns are expected to be non-reproducible. We then applied these three criteria (size, spatial disposition and pattern non-reproducibility) to the description of the 97 embryos. The observed numbers of labelling possibly corresponding to N events (Table 1 right column) strikingly corresponded to those calculated using the fluctuation test (Table 1 left column). This suggests that the above reasoning is correct. The characteristics of the whole library are summarized in Table 2. The number of medium (50 to 100 cells) and large labellings (more than 100 cells) on which this study is based are small (4% to 0.1%). In consequence the probability that they derive from two recombination events (see Materials and Methods) is negligible and thus cannot impact our analysis. For instance, the probability that a clone composed of 200 to 400 cells arises from two clones composed of 100 to 200 cells is equal to 1.9.1024 (2 embryos for 10 000 observed) and the probability that it arises from 4 clones of 50 to 100 cells is 2.5.1026 (3 embryos for one million observed). Similarly the probability that a clone composed of 400 to 800 cells arises from two clones composed of 200 to 400 cells is equal to 1.2.1025 (1 embryo in 100 000 observed). GICL and the SE lox-LacZ library The method of production of labelling whose birth date can be controlled is based on Cre recombinases [29,30], the activity of which depends of a conformational change induced by 4- hydroxytamoxifen (4-OHT) [31]. This approach is generally combined with tissue-specific expression of the recombinase for somatic mutagenesis [32] and GIFM [25]. In GIFM, the aim is to N 0 1 2 3 4 35 (36%) 15 (15%) 4 (4%) 1 (1%) SE clones are classified according to the number of cells they contain. The frequency of large clones was determined in the entire library. The frequency of small clones was determined with a sample of 97 embryos. doi:10.1371/journal.pone.0004353.t002 PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4353 The validity of the LaacZ library was assessed by determining the number of embryos totally lacking SE labelling on a sample of 97 embryos. From this number (42) the number of embryos presenting N events of recombination (from 1 to 4, left column) was calculated using the fluctuation test of Luria and Delbru¨ ck (see Materials and Methods). The right column reports the observed numbers of embryos possibly corresponding to N events (see text for details). doi:10.1371/journal.pone.0004353.t001 induce genetic deletion in the highest number of cells at a given stage of development. In GICL the aim is to induce labelling in a single cell at a given stage of development. Here we report the first use of ubiquitous GICL to analyse SE, a large structure of the embryo. Ubiquitous GICL presents four major specific constraints: 1) every single cell of the animal must express the recombinase, thus making possible the induction of clones in any structure and at any time during development; 2) the recombined reporter gene must be expressed in every descendant of the recombined cell, thus allowing the detection of labelled cell descendants at any selected stage of observation; to fulfil these two criteria, we used inducible ROSA26-driven Cre recombinases and a ROSA26 LacZ reporter gene [26]; 3) the conditional reporter line must be immune to spontaneous recombination that could activate the reporter gene in the absence of the Cre recombinase. ROSA26 LacZ reporter line (R26R) fulfils this criterion as no spontaneous recombination was ever found in R26R embryos and animals (E. Legue´, unpublished data); 4) ideally, the inducible Cre recombinase must have no activity in absence of the inducer molecule; in practice, Table 2. The LaacZ library: size composition of the clones. No of embryos with labelling composed of n cells Entire library No of embryos with labelling composed of n cells Sample of 97 embryos No of cells n .800 400,n,800 200,n,400 100,n,200 50,n,100 20,n,50 10,n,20 ,10 Total no of embryos 0.1% (6) 0.4% (16) 0.4% (15) 1.4% (59) 4% (165) nd nd nd 4248 42 (43%) 30 (31%) 15 (15%) 9 (9%) 1 (1%) 97 0% (0) 0% (0) 1% (1) 3% (3) 7% (7) 12% (12) 21% (20) 51% (49) 97