Supplementary Figure 5: Extra-embryonic ectoderm rosettes facilitate cavities extension and fusion.

(a) Representative example of E5.5 (stage III) embryo stained for E-Cadherin. White dots label the cells contributing to the epiblast (EPI) cavity’s proximal rosette (adjacent to EPI-ExE boundary). Image representative of 15 embryos. (b) Example of a resolving EPI cavity’s proximal rosette (dots show cells contributing to the rosette) through loss of cell-cell contacts (cells marked with hollow dots) of ExE cells at the boundary. YZ projection is displayed on the right of the image. Image representative of 10 embryos (c) Stills from a time lapse movie of an E5.5 LifeAct-GFP embryo showing EPI cavity expansion after EPI cavity’s proximal rosette resolution. Cells are arbitrarily color-coded for tracking. Image representative of 3 embryos (d)E5.5 (stage III) embryo stained for E-Cadherin and tight junction protein ZO-1. Two different z slices are presented (Z0 and Z17, Z step=0.8 um) one showing the ExE cavity and the other the overlaying rosette (white dots). YZ projection of rosette (white dots showing cells contributing to rosette) and ExE cavities shown on the far right of the panel. *=ExE cavity, **=EPI cavity. Image representative of 10 embryos. (e) Representative example of ExE cavity (asterisk in YZ projections, right panel) connected with the centre of an ExE cavity proximal rosette (arrowhead points to the centre of this rosette; magenta dots indicate cells contributing to both rosette and ExE cavity where white dots indicate the rest of rosette’s cells) through a tract of ZO-1 (arrow). White dotted line = EPI-ExE boundary. *=ExE cavity, **=EPI cavity. Image representative of 10 embryos. (f) Two representative examples of an invading EPI cavity and an extended ExE cavity connected with a polarized tract of ZO-1 (white circle). Image representative of 6 embryos. Scale bars = 20um.