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Figure 6. Fibronectin is required for vegetal endoderm cell migration.(A) Endoderm cell morphology on different substrates. Cells on plastic coated with FN (left) are multipolar (red arrows). Cells on gelatin coated with FN (right) are unipolar with front (yellow arrow) and rear (blue arrow). (B) Morphology in vivo. Endoderm cell with front (yellow arrow) and rear (blue arrow) polarity. Animal (An) is up, vegetal (Vg) is down. (C) Cell spreading on FN. Cell shape changes (top row) are outlined (bottom row), consecutive shapes (grey) differ from shapes at previous time points (difference in black). Time in minutes is indicated. (D) Cell locomotion on gelatin-FN in vitro. Morphological changes (top row) are outlined (mid row) along with movement direction (black arrows) and differences in cell shape (black) between time points. A representative cell is followed, panels show bursts of movement over the course of 45 min. Interpretation of cell behaviours (bottom row). (E) Cell migration velocity in vitro. Velocities of cells in explants with respect to the epithelium, and of single cells with respect to in vitro substrates. (F) Inhibition of FN binding in embryos. Embryos were injected with RGD (left) or RGE peptides (center) into the blastocoel at blastula stage 8, or left uninjected (right), cultured until stage 11, fixed, and sectioned sagittally. RGD treatment perturbed endoderm morphology relative to controls (yellow arrows). (G) Inhibition of FN binding in explants. Explants in medium containing RGD (left), or RGE (right) peptides. RGD-treated cells appear rotund (pink outline), RGE-treated cells elongated (yellow outline). Region of interest (red box) is indicated in the top right corner of select panels. (H) Cell length-width ratio in explants incubated in RGE (left) or RGD peptides (right). (I) Cell elongation congruity (defined in Figure 6—figure supplement 1) in explants incubated in RGE (left) or RGD peptides (right). (J) Cell migration velocity in explants incubated in RGE (left) or RGD peptides (right). For H–J, plots show data sampled from three embryos from different egg batches.10.7554/eLife.27190.020Figure 6—source data 1. Quantification of cell migration velocity and congruity.Figure 6—figure supplement 1. Schematic of morphometric analyses.To characterize cell morphology, we analyzed time-lapse recordings of membrane-labelled cells in explants, and in the embryo. For computer-assistant analysis, we first used cell segmentation to identify cell outlines. Segmentation accuracy was manually verified. To find the long-axis of cells, we used the best-fit ellipse to identify the major (a) and minor (b) axes of cells. The cell aspect ratio (a/b) was used to measure the cell body length-width ratio. The long (major) axis of cells were used to determine cell orientation (α) with respect to the animal (An) – vegetal (Vg) axis. To measure the congruence (y/x) of cell elongation relative to the direction of cell migration (along the A-V axis) we measured the corresponding horizontal (x) and vertical (y) cell axes. Image published in: Wen JW and Winklbauer R (2017) © 2017, Wen et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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