Fig. S1. Endothelial expression of Lifeact-EGFP in Tg(Fli1ep:Lifeact-EGFP) transgenic fish. (A) Maximal projection of a confocal z-stack of the head at 3.5 dpf highlighting the subintestinal vein (SIV), aortic arches (AAs) and ventral aorta (VA). Lateral view, anterior to the left. (B) 3D projection of the developing hindbrain vasculature at 53 hpf. Dorsal view, anterior to the top. CtA, central artery; PHBC, primordial hindbrain channel; BA, basilar artery. (C) Endothelial tip cell of a vascular sprout in the boxed region in B. Scale bar: 10 µm. (D) Dorsal view of the head at 3.5 dpf. Anterior to the top. MsV, mesencephalic vein; MtA, metencephalic artery; MCeV, middle cerebral vein; DLV, dorsal longitudinal vein; DMJ, dorsal midline junction; PCeV, posterior caudal cerebral vein; TCB, trans-choroid plexus branch. (E) High magnification of the TCB from D, illustrating junctional complexity of the plexus. (F) Zebrafish trunk at 3.5 dpf. The dorsal aorta (DA), intersegmental vessels (ISVs), dorsal longitudinal anastomotic vessel (DLAV), posterior cardinal vein (PCV), caudal vein (CV), subintestinal artery (SIA) and subintestinal vein (SIV) strongly express Lifeact-EGFP. (G) High magnification of the posterior end of the caudal vein plexus at 4 dpf. Intercellular junctions are distinct in both CA and CV. Scale bar: 10 µm. (H) High magnification of DLAV, arterial ISV (aISV), intersegmental lymphatic vessel (ISLV), DA, PCV and SIA at 3 dpf. Scale bar: 20 µm. (I) ISVs from 2 dpf Tg(Fli1ep:Lifeact-EGFP) embryos stained with phalloidin (magenta). Scale bar: 10 µm. (J) High magnification of boxed region in I, showing colocalisation of Lifeact-EGFP with phalloidin in an ISV (arrows).

Fig. S2. Actin filaments form at cell junctions. Still images from a movie of an embryo with clonal expression of Lifeact-EGFP and mCherry-hZO1 in ISVs and DLAV. F-actin (Lifeact) is formed at ZO1-positive cell junctions (arrowheads) and undergoes similar rearrangements as ZO1 as the ISV and DLAV become lumenised. Scale bar: 10 µm.

Fig. S3. F-actin dynamics in filopodia. (A,B) Length of Lifeact-EGFP and mCherryCAAX signal during the lifetime of two filopodia. Filopodia at the leading edge of endothelial tip cells from 30 hpf Tg(Fli1ep:Lifeact-EGFP); Tg(Kdr-l:ras-Cherry)s916 embryos were analysed.

Fig. S4. Latrunculin B disrupts F-actin polymerisation and endothelial cell morphology. Tg(Fli1ep:Lifeact-EGFP); Tg(Kdr-l:ras-C herry)s916 28.5 hpf embryos were treated with 2 µg/ml Lat. B. (A) At 20 minutes post-addition of Lat. B, endothelial tip cells of ISVs display multiple F-actin-filled filopodia (arrows) and junctional F-actin at the dorsal aorta (white arrows). (B-D) As drug treatment continued, fewer filopodia protrusions are observed. There is an increase in F-actin-positive puncta (black arrowheads) and loss of junctional F-actin (white arrowhead in D). Concomitant to perturbation of F-actin polymerisation, there is a change in EC morphology. The cell membrane becomes more convoluted at the cell body and at cell protrusions (magenta arrows). (E) Number of CAAX-positive filopodia after addition of 2 µg/ml Lat. B. n=2 ISVs from two embryos.

Fig. S5. Endothelial cells migrate at different velocities during ISV formation. (A) Velocity profile of one tip cell nucleus during ISV migration. Dashed lines separate the myoseptum into three regions: below, at, and above. (B) Plot of tip cell nuclei velocities below, at and above the horizontal myoseptum during ISV migration. Mean ± s.d. Below, n=173; at, n=274; above, n=629. Number of tip cells: below, 7; at, 8; above, 9. Number of embryos, 3.

Fig. S6. Endothelial cells of vein plexus are dependent on filopodia for anastomosis. Tg(Kdr-l:ras-Cherry)s916 embryos were treated with (A) 0.4% DMSO or (B) 0.1 µg/ml Lat. B from 22 hpf and imaged at 55 hpf. Vascular sprouts of the vein plexus become globular in morphology and fail to undergo anastomosis to form the ventral vein. DA, dorsal aorta; DV, dorsal vein; VV, ventral vein. Scale bars: 10 µm.

Movie 1. F-actin dynamics during cell division. ISV of 32 hpf Tg(Fli1ep:Lifeact-EGFP) embryo displaying dynamic F-actin polymerisation during cell division.

Movie 2. Dynamic F-actin polymerisation in filopodia during endothelial cell migration. A migrating ISV of 31 hpf Tg(Fli1ep:Lifeact-EGFP) embryo showing dynamic F-actin polymerisation in filopodia. Lamellipodia-like protrusions emerge from the most persistent filopodia.

Movie 3. Endothelial cells without filopodia migrate in a guided manner. Tg(Fli1ep:Lifeact-EGFP) embryos were treated with 0.4% DMSO or 0.1 µg/ml Lat. B from 24 hpf. Time-lapse imaging began at 30 hpf.

Movie 4. Low Lat. B concentration decreases endothelial cell migration velocity. Tg(Fli1a:nEGFP) embryos were treated with 0.4% DMSO or 0.1 µg/ml Lat. B and imaged 30 minutes later.

Movie 5. Endothelial cells migrate with lamellipodia in the absence of filopodia. ISV migration in Tg(Fli1ep:Lifeact-EGFP) embryos pre-treated with 0.08 µg/ml Lat. B. Time after addition of drug is indicated. Arrow indicates formation of contractile ring.

Movie 6. Endothelial cells lacking filopodia respond to ectopic Vegfa165 cues to form misguided vessels. Tg(Kdr-l:ras-Cherry)s916 embryos injected with HSP:Vegfa165-IRES-nlsEGFP plasmid were heat shocked at 25.5 hpf and treated with 0.08 µg/ml Lat. B from 29.5 hpf. Time-lapse imaging began at 31.5 hpf. Vegfa165-expressing cells are labelled with nuclear EGFP. mCherryCAAX is magenta. Arrows point to ectopic vessel sprouts.

Movie 7. ISV, DLAV and vein plexus formation in control and Lat. B-treated embryos. Tg(Fli1ep:Lifeact-EGFP) embryos were treated with 0.4% DMSO or 0.1 µg/ml Lat. B and imaged 1 hour later at 30 hpf.