Supplementary Figure 1: Fibronectin and interstitial fluid localization at the neurectoderm-to-prechordal plate interface during zebrafish gastrulation.

(a–c) Immunofluorescence confocal images of the neurectoderm (ecto)-to-prechordal plate (ppl) interface (white dashed line) in a wild type (wt) embryos at 6 (a), 8 (b), and 9 (c) hpf showing Fibronectin staining (pseudo-colored with Fire LUT) in maximum intens ity projections of dorsal views (top panels) and sagittal sections (middle panels); red dashed line outlines position of ppl leading edge cells; blue dashed line indicates ecto-to-EVL interface, and yellow dashed line shows YSL interface to ppl and ecto; bottom panels are sagittal sections of the ecto-to-ppl interface stained for F-actin (phalloidin) to mark this interface; double-sided arrows indicate animal (A) to vegetal (V) and dorsal (V) to ventral (V) embryo axes; asterisk labels ppl leading edge cell; Scale bar, 20 μm. (d) Multiphoton live cell images showing interstitial fluid (IF) accumulation (dextran-Alexa Fluor 647, left panel), F-actin localization (Tg(actb1:lifeact-GFP), middle panel) and a combination of those different labels (right panel) at the ecto-to-ppl interface (white dashed line) at 7 hpf; red arrows indicate extracellular cavities filled with IF at the ecto-to-ppl and ecto-to-YSL interfaces; white arrows indicate ecto-to-ppl cell-cell contacts devoid of IF accumulations; blue dashed line indicates ecto-to-EVL interface, and yellow dashed line shows YSL interface to ppl and ecto; double-sided arrows indicate AV and dorsal DV embryo axes; asterisk labels ppl leading edge cell; Scale bar, 20 μm. (e) Multiphoton live cell image of Tg(gsc:GFP) embryo (t = 120 min, 8 hpf) with pseudo-colored spots marking positions of nuclei within the axial mesendoderm (green); dorsal view with double-sided arrows indicating AP to VP and left (L) to right (R) embryo axes; color-code indicates mean total cell speeds of axial mesendoderm cells moving to the animal pole after internalization (cyan, 0–2 and yellow/magenta >2 μm min⁻¹); position of anterior (ppl) and posterior mesendoderm marked; Scale bar, 50 μm. (f) Average instantaneous cell speeds in μm min⁻¹ of internalized axial mesendoderm cells in wt embryos (n = 6 embryos) plotted along the normalized distance along the AV axis from anterior (0) to posterior (1); green dashed line marks position of transition from anterior (ppl) to posterior axial mesendoderm, error bars, s.e.m.