Movie 8

Anastomosis in an esama^ubs19; ve-cad^ubs8 double mutant embryo
Deconvolved spinning disc confocal movie of a Tg(fli1ep:gal4ff)^ubs3, Tg(UAS:EGFP-UCHD)^ubs18, Tg(kdrl:mCherry-CAAX)^s916 esama^ubs19; ve-cad^ubs8 double mutant embryo at around 32hpf, anterior to the left. Confocal stacks were acquired every minute. Single channels are shown in inversed contrast (green is EGFP-UCHD and red is mCherry-CAAX on left and right, respectively) and the merged channels are shown in the middle. As the two tip cells migrate towards each other, they probe the environment for neighboring tip cells. Filopodia of the adjacent tip cells touch occasionally. At the 59^th minute we observe a filopodial contact between the two tip cells, which appears to be reinforced by the actin cytoskeleton, but is resolved shortly after. As the two cell bodies get closer to each other, they start to touch and retract again. This happens several times and even when the two cell bodies overlap extensively (see 97^th minute), they resolve the contact made. Eventually, the two tip cells appear to form a cell-cell contact and to maintain it (around the 201^st minute of the movie). We imaged this embryo over 3h and anastomosis takes significantly longer compared to wild-type and even ve-cad^ubs8 mutant embryos. In the last 20 minutes of the movie, we observe junction-like patterns with the EGPF-UCHD marker. However, in many other instances, we observed that some tip cells remain isolated, even after they tried to establish contact to neighbors.