Scale bars are 50µm.Įxtensive migratory events, morphological changes, and cell divisions coordinated by cell signaling drive complex vertebrate patterning events during embryonic development as well as during epimorphic tissue regeneration. ( C) Isolated zoomed images of the segmented nuclei and membranes during the time-lapse. ( B) Segmented and photoconverted memb-Dendra2 (red) as well as the segmented H2B-Cerulean (multi-colored surfaces) along with the merged animal pole images as seen in Video S3D. Note that the developing head and optic primordia can be seen during the time-lapse. ( A) Segmented and photoconverted memb-Dendra2 (red) as well as the segmented H2B-Cerulean channel (multi-colored surfaces) along with the bright field (grayscale) from the time-lapse video. The nuclei were tracked starting with the final frame, moving backward in time. Then, using the segmented data, nuclei within the surrounding photoconverted membranes were identified and represented as colored surfaces. First, the photoconverted membranes were segmented in 3D. This video shows segmented data for each frame of the time-lapse summarized in Figure 2 in the text. Video S4: Segmented Time-Lapse Data for PhOTO-M Membrane Dynamics Visualization During Late Gastrulation. Note that the epiblast cells move beneath the field of view in the last frame, due to the development of the head during early segmentation.

Each cell undergoes a single cell division during the course of the time-lapse. Two enveloping layer (EVL) cells are photoconverted (the orange and red spheres) as well as an epiblast cell (the white sphere). ( E) Segmented nuclei from the intensity images from panel (D). ( D) Intensity images from segmented photoconverted nuclei (red) for the same 4 timepoints as the merged image in panel (C). ( C) Zoomed in MIP areas at four different time-points of the merged fluorescence images as a reference for the photoconverted images seen in panels (D) and (E). ( B) Merged MIP image of the final time frame of the time-lapse. ( A) Merged MIP of the first frame of the time-lapse, showing memb-Cerulean (blue) and both unconverted (green) and segmented photoconverted (red) H2B-Dendra2. Animal pole view maximum intensity projection (MIP) images of the first 10.5µm (in depth from the animal pole) of an ∼6 hour time-lapse of a heterozygote F1 PhOTO-N zebrafish from late gastrulation (>80% epiboly) until early segmentation.

Figure S2: Nuclear Photoconversion and Segmentation in a PhOTO-N Zebrafish During Gastrulation.

Focus stacking software for photoline