Figure 1. Cdx2-eGFP is an early marker of the developing TE lineage, governed by Hippo signaling differences from the early 16 cell stage.

(A) Immunofluorescence staining against Cdx2 and eGFP in Cdx2-eGFP heterozygous embryos at different stages. Representative images of 10 8 cell, 39 16 cell, 35 32 cell and 11 64 cell embryos stained and imaged in two independent experiments. Scale bar: 25 μm. Correlation between eGFP and endogenous Cdx2 signals was calculated by measuring fluorescence intensities in individual cell nuclei and performing Pearson’s correlation (r indicates coefficient). p-values are also given for each embryonic stage. (B) Mean fluorescence intensity of eGFP (Y-axis) in individual inside and outside cell nuclei of different stage Cdx2-eGFP embryos. Position was determined by co-staining embryos with phalloidin (F-actin) and cells with any surface membrane exposure were classified as outside. n indicates number of embryos. * and ** note how eGFP/Dapi measurements segregate in individual embryos. Statistical significance was calculated by Mann-Whitney test and significant p-values are indicated. Error bars: s.d. of mean. (C) Mean eGFP intensity relative to nuclear/cytoplasmic Yap ratio in individual inside (red) and outside (blue) cells in Cdx2-eGFP embryos at different stages. Representative measurements from 5 8 cell, 8 early 16 cell, 5 late 16 cell, 5 early 32 cell and 4 late 32 cell embryos are shown. All embryos were stained and imaged in one experiment. Correlation was calculated using Pearson’s correlation (r indicates correlation coefficient) and p-value is given. (D–E) Mean fluorescenceintensity of eGFP (Y-axis) in single cells in different cell populations, in early and late 16 cell stage Cdx2-eGFP embryos. (D) Inside apolar, outside apolar and outside polar cell populations. (E) Inside cells, outside cells with low nuclear/cytoplasmic Yap ratio and outside cells with high nuclear/cytoplasmic Yap ratio. Polarity was determined by phospho-ezrin staining. n indicates number of embryos analyzed. Statistical significance was calculated by Kruskal-Wallis test and significant p-values are indicated. Error bars: s.d. of mean. Cells in M-phase are not included.

DOI: http://dx.doi.org/10.7554/eLife.22906.003

Figure 1—figure supplement 1. Developmental staging of Cdx2-eGFP embryos.

Cdx2-eGFP embryos were always staged based on cell number, which we determined in live embryos based on the number of Cdx2-eGFP positive cells present. An additional layer of ‘early’ and ‘late’ sub-staging was included, which refers to the time of embryo isolation. For example ‘early 16 cell’ embryos were harvested at E2.5 – a time point when the population of embryos are between 8 and 16 cell stages – but only 16 cell embryos were used (embryos with average of 12 visible Cdx2-eGFP positive cells). Or ‘late 16 cell’ embryos were harvested at E2.75 -when embryos are between 16- and 32 cells - however only strictly 16 cell embryos (embryos with average of 12 visible Cdx2-eGFP positive cells) were used from this time point. We established criteria for staging using the number of Cdx2-eGFP positive cells in live embryos. Graph above shows average number of Cdx2-eGFP positive cells in live staged embryos at each stage (8 cell n = 10, early 16 cell n = 14, late 16 cell n = 19, early 32 cell n = 21, late 32 cell n = 24, ~64 cell n = 11 and ~80 cell n = 14). A subset of staged embryos were fixed and total cell numbers were determined by Dapi staining (8 cell n = 10, early 16 cell n = 14, late 16 cell n = 15, early 32 cell n = 21, late 32 cell n = 24, ~64 cell n = 11 and ~80 cell n = 11). Error bars indicate standard deviation of mean. Using this guide, only carefully staged embryos were used at each time point for all experiments in the study.

DOI: http://dx.doi.org/10.7554/eLife.22906.004