Figure 2.

A wave of gradual cell polarization traverses the epiblast before primitive streak formation. (a–c) Analysis of the AR between the major and minor axis of epiblast cells measured from scanning electron micrographs represented as a heat map. AO (blue brackets), hypoblast (hypo, light green arrowheads) and KS (cyan arrowheads) are indicated. (d–e) Quantification of average AR in the AP and AO shows an increase of AR in the AP (d) with development, while no such change is seen in the AO (e). Mean ± s.e.m. shown; one-way ANOVA followed by Tukey's post-test: n.s., not significant; a–c indicate significant difference at p < 0.0001 (further details in electronic supplementary material, table S2). (f–i) AR of anterior (ant) and posterior (pos) regions compared at EGK X and EGK XIII. (j–k) AR between the two regions at EGK X and XIII. No significant difference at EGK X (j), but significantly higher AR in the posterior region at EGK XIII (K). **** p < 0.0001; unpaired t-test (further details in electronic supplementary material, table S2). (l–t) Polarity markers show progressive apical localization during development. PAR3 and PKCζ are first concentrated in nuclei. The Golgi marker GM130 becomes localized from EGK XIII (s–t). Phalloidin staining for f-actin marks cell boundaries. (u–w) The number of microvilli on the apical surface (arrows) increases gradually during development. (x–ai) Rho family small GTPases RAC1 and RHOA gradually increase in expression and become concentrated posteriorly with development. (aj–ak) Ventrally, SEM shows the gradual formation of the hypoblast sheet (aj–aj″ and ak–ak″). At EGK X–XI, posterior cells are interconnected (aj″), whereas anterior cells are dispersed (aj′). At EGK XII, the hypoblast sheet covers half of the AP diameter (ak). Higher magnification (ak′, ak′) shows numerous filopodia and lamellipodia. Scale bars: 100 µm in (a–c), (f–i), (l), (m), (o), (p), (r), (s), (x–ai), (aj′), (ak′); 10 µm in (n), (q), (t), (aj″), (ak″); 2 µm in (u–w).