Supplementary Figure 4: Anterior-posterior patterning in ETX embryos.

a. Images showing the same ETX embryos presented in Fig.3a-b with intensity profiles for Nodal HBE-YFP fluorescence. Insets show intensity of YFP signal in embryonic compartment. White boxes indicate area selected for intensity measurement (right-most graphs). Orange line indicates midline of the structure. Surface plot graphs (middle) show intensity of YFP. Bar=20μm. b. Proportion of structures expressing either asymmetric or symmetric Nodal at day 4 versus day 5. n=20 structures per group, 3 experiments. c. Quantitative assessment of endogenous Nodal HBE-YFP asymmetric fluorescence intensity in representative ETX embryos at day 4 (left) and day 5 (right) presented in (a). Each dot represents a cell. Mean intensity was calculated for cells in the region left or right of the midline. Two-sided Student’s t-test, n=83 (left of the midline), n=81 (right of the midline) cells in day 4 ETX embryos; n=153 (left of the midline), n=159 (right of the midline) cells in day 5 ETX embryos. Means ± SD. d. Whole mount in situ hybridization revealing Cerberus (3 embryos in 3 experiments; 8 ETX embryos in 3 experiments), Nodal (4 embryos in 2 experiments; 10 ETX embryos in 3 experiments), T/Brachyury (3 embryos in 2 experiments; 17 ETX embryos in 3 experiments), Cripto (7 embryos in 2 experiments; 13 ETX embryos in 2 experiments), Wnt3 (9 embryos in 3 experiments; 10 ETX embryos in 3 experiments) and Bmp4 (8 embryos in 2 experiments; 14 ETX embryos in 2 experiments) transcripts in natural embryos and ETX embryos at indicated time points. Bar=50μm.