Double fluorescence time-lapse frames of QH1-positive cells and Mito-GFP labeled cells.

<p>All GFP-labeled cells were derived from electroporated intra-embryonic mesoderm shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone-0060841-g004" target="_blank">Figure 4</a>. This figure is comprised of a series of image frames from a time-lapse recording of the embryo in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s007" target="_blank">Movie S6</a> (i.e., Fig. 5, Fig. 6 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s007" target="_blank">Movie S6</a> are all the same specimen). Time points and regions of interest (ROI, white boxes) were selected to denote salient features. Each horizontal strip shows five images depicting four time points. The first and second frames in each strip depict the same time point acquired in both the red (QH1) and the green (GFP) optical channels, respectively (e.g., frames 36′ and frame 36). The succeeding three frames in the row show the Mito-GFP signal only. The white arrows denote QH1 positive cells in frame 36′ that correspond to the Mito-GFP positive cells in frame 36. During the time-lapse recording primordial endothelial cells shifted positions and engaged in cell division shown in frames 36-47. For example by frame 41, the second cell from the top (ROI) is condensed and rounded, suggesting possible cell replication, followed by cellular spreading (frames 42, 47). By the time frame 66 was captured all of the Mito-GFP cells in the ROI at frame 52 had moved and/or rounded up. Also, compared to earlier time points fluorescent cells in frame 66 are reduced in number indicating that one or more cells had departed the ROI. After frame 61 the cells of interest continued to move apart, compared to earlier time points; this is denoted by a larger ROI (white box). The GFP-labeled cells visible in frame 70 (circles) are QH1-positive (white boxes with arrows frame 70′). The encircled cell at the bottom of frame 70 is absent from the ROI approximately 13 minutes later (frame 73). In the interval between frames 79 and 80 a cell of interest (circle) was lost from its previous position. The same behavior is observed between frames 84 to 100. The QH1/GFP-positive cells are progressively lost from the ROI, such that by frame 100 all the original cells of interest (frame 36) are absent. Note the consistently bright cells at the periphery of the ROI; this shows that electroporated mesoderm maintains robust Mito-GFP expression throughout the recording. See Methods and Materials, and the Legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s007" target="_blank">Movie S6</a> regarding details on optics and image acquisition. Scale bar = 100 µm.