ECs are required to induce pericyte motility and proliferation in 3D collagen matrices during EC-pericyte tube coassembly. Nuclear GFP–labeled pericytes (nuc-pericytes) were incorporated into 3D collagen matrices in the presence or absence of ECs and allowed to undergo morphogenesis over a period of 120 hours. Real-time video analysis was performed to assess pericyte motility and proliferative rates over 0-72 hours or 72-120 hours. (A) Representative images of tracking analysis that were done with nuc-pericytes over 0-72 or 72-120 hours demonstrates the requirement of ECs to induce pericyte motility in 3D matrices. (B-C) From the tracking data generated in A, the average distance pericytes moved from their point of origin and the average pericyte velocity was measured for both 0-72 hours (B) or 72-120 hours (C). Both of these measures display impaired pericyte motility and decreased pericyte velocity over time. Furthermore, in the absence of ECs, the pericyte proliferative rate is reduced over both time periods, as quantified by the total number of pericytes per high-powered field over time (B-C). n ≥ 5; P ≤ .01.