Fig. S4

Analyses of pruning vessel with the cell membrane marker.

The TgBAC(kdrl:mKate-CAAX) transgenic line allows visualization of the endothelial cell membranes. Transcellular lumen formation and collapse can be distinguished by the presence of inflated, round-ended, apical membrane compartments (black arrows). In certain cases, it is also possible to recognize multicellular vessel fragments by the increase in staining density at the cell–cell contact surfaces (green arrows). The nuclei are recognizable as round structures that are brighter because the two membranes (apical and basal) are well separated around the nucleus (red asterisks in the vessels of interest). These criteria were used for quantifications presented in Fig 3C. Three pruning vessel segments are followed in this figure. Segment 1: lumen in a unicellular vessel fragment (black arrow) is parted next to the nucleus (red asterisk). The lumen perfuses (B) and deflates again (C) to finally collapse completely (D) when the vessel detaches (E, dotted line). Segment 2: a multicellular vessel fragment with the cell–cell contacts visible as darker lines of the membrane staining (A–D, green arrows) narrows significantly when the lumen collapses (E–F). Cells leave the branch after the lumen collapse (D–F, asterisks mark the nuclei). Segment 3: a unicellular vessel fragment undergoes cell division generating two nuclei (C, asterisks), a new cell–cell contact surface (C, green arrow), and two luminal compartments (C, black arrows). The lumen is partially restored (D) but later collapses, leaving a small vacuole-like structure (E, black arrow). The last cell–cell contact surface is reduced as the pruning proceeds (E–F). See also S11 Movie.