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Supplementary figures

Supplementary figure 1: In vivo analysis of Wnt8a localisation in PAC2 fish fibroblasts and in

epiblast cells in zebrafish.

a, Wnt8a antibody staining of primary cell culture from marginal cells or from animal cells from

zebrafish embryos at the 50% epiboly stage. Endogenous Wnt8a protein can be detected in fixed

marginal cells at the tips of cell protrusions (arrow), whereas animal pole cells are Wnt8a-negative b,

Live imaging of clones of epiblast cells expressing palmitoylated GFP (Palm-GFP) or GPI-anchored

GFP (GPI-GFP) in zebrafish at 50% epiboly. c, Fgf8a-GFP is visible in the extracellular space, and

accumulates in membrane clusters and in endosomes of receiving cells. d, In contrast, Wnt8a-GFP is

strongly associated with membranes of the producing cells (arrows) in zebrafish embryos. e, Live

imaging of PAC2 cells after transfection with LifeAct-GFP and Wnt8a-mCherry. f, Triple expression

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of memCFP together with Toca1-GFP and Wnt8a-mCherry in clones in zebrafish embryos. Arrow

points to co-localisation of Wnt8a and Toca1 at filopodia nucleation points. All scale bars are 10µm.

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Supplementary figure 2: Measurement of filopodia length in epiblast cells during zebrafish

gastrulation and PAC2 fibroblasts.

Zebrafish embryos were injected with membrane marker in one blastomere at the 8 cell-stage for

analyses of the length of cell protrusions. a, At 50% epiboly, embryos were subjected to high-

resolution laser-scanning confocal microscopy. After scanning, 3D stacks were assembled to analyse

morphology of single epiblast cells. Image post-processing included a representative 3D surface

rendering. Cell protrusions were identified automatically by using the FILAMENT TRACER module

of Imaris 7.1. To identify filopodia, the following criteria were chosen: starting point (largest

diameter, e.g., base of filament) 1.5 µm; seed point (thinnest diameter, e.g., dendrite ending) 0.4 µm;

distance for connection (shortest distance from distance map) 3.2 µm. All filopodia were manually

validated. b, In vivo imaging of PAC2 cells transfected with Wnt8a-GFP and Evi-mCherry revealed

cell protrusions with lengths up to 70 µm in-vitro in a cell culture dish.

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Supplementary Figure 3: Activation of the Wnt signal transduction cascade in co-cultivated

PAC2 cells. a, PAC2 cells transfected with Wnt8a-GFP were co-cultivated with PAC2 cells

transfected with Dvl2-mCherry. Wnt8a located on filopodia tips can recruit Dvl2 to the membrane.

Inset is shown in high magnification and after 3D surface rendering. b, Co-transfection of Evi

increases the membrane presentation of Wnt8a-GFP in PAC2 cells (blue arrows). c, Evi co-

transfection increases the activation of the SuperTopFlash (STF) reporter expression in a HEK293T-

cell-based co-cultivation assay. Graph shows relative activation in the TOPFlash reporter assay. Error

bars represent S.E.M.

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Supplementary Figure 4: Analysis of Cdc42 function and its influence on Wnt8a secretion.

a, Confocal images of PAC2 fibroblasts transfected with Wnt8a/Gap43:GFP together with Cdc42WT or

Cdc42T17N. Fibroblasts transfected with full-length Cdc42 displayed a 2.0 +/- 0.5-fold increase in the

number of filopodia per cell compared to control (n=15). Consistently, transfection with the dominant-

negative Cdc42T17N showed a 3.3 +/- 0.2-fold reduction in the number of filopodia (n=15) b, Confocal

images of pmKate2-positive mouse 3T3 control cells and cells transfected with either Cdc42WT-GFP

or Cdc42T17N-GFP. The number of filopodia present in cells after transfection with Cdc42 is strongly

increased compared to control cells. Filopodia number is reduced after transfection of the dominant

negative form, Cdc42T17N. Insets show parts of the cell membrane at higher magnification. c, Live

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imaging of cell clones expressing Wnt8a/mCherry-GPI and Cdc42WT or Cdc42T17N in a zebrafish

embryo expressing Lrp6-GFP at 50% epiboly. Embryos carrying a Wnt8a/Cherry-GPI clone were

treated with 0.25 nm Latrunculin B from 3–6 hpf prior imaging. Lrp6-GFP expression in host cells

and Wnt8a-mCherry expression together with the expression of mCFP in donor clone cells are

displayed. Yellow arrows point to co-localisation shown in Fig. 4f.

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Supplementary Figure 5: Filopodia activity and anteroposterior patterning in the neural plate in

zebrafish.

a, Classification of phenotypes according to their expression of the axin2 and otx2. b, Quantification

of the expression levels of cdc42 by qRT-PCR. At the one cell stage, embryos were injected with

Wnt8a mRNA, 0,2 ng.. Total mRNA was isolated from 50 embryos at 50% epiboly. This experiment

was conducted in triplicate. c, Expression of axin2 in embryos at 50% epiboly treated as indicated.

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Supplementary Figure 6: Simulation of ligand concentration in a morphogenetic field based on a

filopodia-based distribution mechanism.

a, Schematic drawing of the expansion of the neural plate in zebrafish from 50 µm at 4.6 hpf to 640

µm at 8 hpf. Lateral view of the entire embryo and dorsal view of the neural plate, with lengths

indicated. b, Complete list of implemented parameters used during the simulation. c, Schematic

representation of neural plate expansion by intercalation. c, During epiboly movement, neural plate

cells (pink) intercalate (red arrows) leading to an expansion (green arrows) of the neural plate (left).

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Wnt8a-positive source cells (blue) stay at the posterior end of the plate and do not intermingle with

neural plate cells. (Right) During each intercalation step cells migrate into the neural plate. This leads

to a displacement of neighbouring cells until a free field (empty ring) is reached. Effectively, the

shortest path between these two points is taken. The Wnt8a activity status of each cell on this path is

then translated in a cascade from the starting point (red) to the final empty cell field (purple). The

migrated cell is marked by an x. d, Snapshot of two simulation steps showing Wnt8a-producing cells

(blue) and Wnt8a-receiving cells: Wnt8a high (dark red), Wnt8a low (light red) and empty cell fields

(light blue rings) at consecutive time points. e, Simulation results with identical contact frequency for

filopodia lengths 11.9 +/- 2.9 µm, and 23.8 +/- 10.6 µm. No difference in the anteroposterior position

of the midbrain-hindbrain boundary (MHB) was observed.