Supplementary Figure 2: Temporal Dox administration in Mes1-rtTA/TetO-Cre/Rosa-Confetti embryos.

(a) While clonal dose of DOX (0.575 μg g⁻¹) induces labelling in Mesp1-rtTA/TetO-Cre/Rosa-Confetti embryos at E6.25 (n = 53), at E6.75 (n = 118) or at E7.25 (n = 65), this dose was not sufficient to induce labelling at E5.75 (n = 13). A much higher dose of Dox (25 μg g⁻¹) was required to produce labelling at a clonal density at E5.75 (n = 90). This 40 fold increase of DOX is likely to persist at a concentration sufficient to activate the Cre at the time of endogenous Mesp1 expression. This high dose of DOX never labelled any heart after administration at E8.5 or E9.5 (n = 24) supporting the absence of transgene expression after the end of endogenous Mesp1 expression. (b,c) Examples of Mesp1-rtTA/TetO-Cre/Rosa-Confetti unicolour labelled hearts at E12.5 induced at E5.75 after administration of high dose of Doxycycline (25 μg g⁻¹). Note that each cluster is localized within the LV, FHF derivative and no labelling was detected other compartments. OFT, outflow tract; RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; IFT, inflow tract. Scale bars, 200 μm. The number on the upper right in each panel refers to the ID of the labelled heart. (d) Quantification of the regional (FHF and SHF) contribution of patches of Mesp1 labelled cells in unicolour hearts induced at E5.75 with the high dose of Doxycycline (25 μg g⁻¹), shows the exclusive labelling of the FHF (red) similarly to was found at E6.25 (Fig. 2m).