Z-projection of a timelapse video of Tg(cldnb:lyn-gfp) (green) cells transplanted into Tg(cldnb:lyn-mscarlet) embryos, showing superficial membrane protrusions. Processed Airyscan video.

Two cells of each class (leading, protoneuromast, superficial) are shown. Relative positions in the PLLp are shown in the bottom panel. Enlargements of each cell are shown above from each orthogonal view (corresponding to the colored arrows in the bottom panel).

Two cells of each class (leading, protoneuromast, superficial) are shown. Relative positions in the PLLp are shown in the bottom panel. Enlargements of each cell are shown above from each orthogonal view (corresponding to the colored arrows in the bottom panel).

(A) Top panel: 3D rendering of PLLp. Superficial cells in magenta, protoneuromast cells in green and all other cells in gray. Bottom panels: Cross sections at the positions indicated in A showing internal PLLp cell boundaries in gray, and external cell boundaries in either magenta (for superficial cells) or green (for protoneuromast cells). (B) Boxplot of external (first two columns) and total surface area size (second two columns) for superficial and protoneuromast cells from the segmented PLLp shown in A. p-values are Mann-Whitney nonparametric U test. (C) 3D rendering of all segmented nuclei in the PLLp at t = 0 from Figure 2—video 2A. Box represents all nuclei within 40 µm (lengthwise) of the central protoneuromast in the PLLp, and includes the nuclei tracked in panels B-F. (D–F) Cross-section projections of the boxed area in A (from the perspective of the black arrow), showing each individual nuclear centroid track in a different color over 120 1-min timepoints (2 hr total). (D) Superficial nuclei tracks. (E) Protoneuromast nuclear tracks. (F) All nuclear tracks. (G–I) Heatmaps of the centroid positions of the nuclei in D-F, colored by whether the nucleus belongs to a superficial (magenta) or protoneuromast (green) cell (see panel A). To generate cross-section tracks in D-F, nuclear positions were normalized to the central apical constriction of the protoneuromast (which lies at 0,0 in all of the plots) to correct for migration of the primordium in the (X and Y plane), and a small rotation (~10° counter-clockwise) was imposed to compensate for asymmetries in the time-lapse data acquisition. See also Figure 2—video 2 for more details.

Z-projection of a timelapse video taken adjacent to the skin in TgBAC(cxcr4b:lifeact-citrine) (green); Tg(cldnb:lyn-mscarlet) (magenta) double transgenic embryos, showing superficial protrusive activity. Processed Airyscan video.

Top panel shows control (unmanipulated) and bottom panel shows experimental (skin removed) side of the same embryo. Images are maximum-intensity projected stitched confocal stacks overlaid with DIC images.

Top panel shows control (unmanipulated) and bottom panel shows experimental (skin removed) side of the same embryo. Images are maximum-intensity projected stitched confocal stacks overlaid with DIC images.

Z-projection of a timelapse video of TgBAC(cxcr4b:lifeact-citrine) (green); Tg(cldnb:lyn-mscarlet) (magenta) embryos showing blebbing of superficial cells after skin removal. Processed Airyscan video.

(A) Examples of superficial and basal protrusions in both skin-intact and skin-removed conditions. All protrusions were measured from time-lapse images where the direction of protrusion extension and maximal extension length could be quantified. (B) example kymograph used for quantification of retrograde actin flow. Left panels represent single frames from time-lapse video. Red arrow indicates the position sampled for the kymograph (right).

(A) Examples of superficial and basal protrusions in both skin-intact and skin-removed conditions. All protrusions were measured from time-lapse images where the direction of protrusion extension and maximal extension length could be quantified. (B) example kymograph used for quantification of retrograde actin flow. Left panels represent single frames from time-lapse video. Red arrow indicates the position sampled for the kymograph (right).

(A) Superficial LifeAct-positive protrusions (green) and membrane (magenta) in skin-removed embryos embedded in 20 mg/mL Matrigel. (B) Quantification of superficial protrusion directionality in embryos embedded in Matrigel (C) Basal LifeAct-positive protrusions (green) and membrane (magenta) in skin-removed embryos embedded in 20 mg/mL Matrigel (D) Quantification of basal protrusion directionality in embryos embedded in Matrigel. (E) Box plots of average cell migration speed (in the direction of normal migration) for embryos with skin intact embedded in 1% agarose, skin removed embedded in 1% agarose, skin removed and regrown embedded in 1% agarose, skin intact in 20 mg/mL Matrigel, and skin removed embedded in 20 mg/mL Matrigel. Dots indicate individual cell speed in the direction of normal migration in at least three embryos. p Values are represent results from ANOVA with multilevel model. Panels (A, C) are processed Airyscan super-resolution images.