Time-Lapse Imaging of Asymmetric Spindle Positioning During Endothelial Tip Cell Division in Angiogenesis In Vivo.

The branching of new blood vessels by angiogenesis is critical to the development, growth, and repair of most vertebrate tissues and is frequently dysregulated in disease. At its core, angiogenesis is driven by the collective migration of leading "tip" and follower "stalk" endothelial cells. Recent work reveals that this collective movement is coordinated by asymmetric tip cell divisions that generate daughters of distinct size, signaling capacity and tip-stalk behaviors. Polarized mitotic spindle positioning is critical to generating such asymmetries in daughter cell size. However, the spatiotemporal dynamics of vertebrate spindle movement are often difficult to explore using in vivo systems. Here we describe a method for the sample preparation, live-imaging and data analysis of endothelial cell mitotic spindle positioning in developing zebrafish embryos. This method enables single-cell and population-level spindle dynamics to be monitored and quantified, both in wild-type or genetically/pharmacologically perturbed embryos. Moreover, this approach can be easily adapted for live imaging of spindle dynamics in other zebrafish embryonic tissues that experience similar asymmetric divisions, such as the trunk neural crest.