JBL generate luminal connectivity.

a: High-resolution time-lapse video (video s1) of EGFP-ZO1 and mRuby2-UCHD during junctional rearrangements. Yellow asterisks point to JBL. Yellow doubleheaded arrows indicate the distance between the junction and the dorsal end of the DLAV. b: Plots displaying the signal intensities of EGFP-ZO1 and mRuby2-UCHD, along the white lines, at different time points, during several JBL cycle. c: still images of time-lapse video (video s2) showing EGFP-Podxl1 and mRuby2-UCHD during luminal fusion, starting at approximately 32 minutes. The leading edges of the converging junctions are indicated by white arrowhead. Yellow arrowheads point to JBL. scale bar: 10 μm. d: Graphic depiction of blood vessel lumenization by cell convergence. Top: At first, cells 1 and 3 and cell 2 and 3, respectively, are forming cell-cell interfaces (cell junctions: green), which enclose local lumens (apical membrane: purple). Middle: JBL drive convergent cell movements, which lead to the formation of a novel cell contact (cells 1 and 2) and the merging of the two lumens into one (bottom). Graphic depiction of a lumenized DLAV, displaying the multicellular architecture. e: still images of time-lapse video s3 showing the oscillatory behavior of JBL (yellow arrowhead) during DLAV formation (30hpf). The white dashed line surrounds the UCHD-labeled JBL domain. scale bar: 5μm. A schematic representation of the time-lapse video is shown in the bottom panel.

JBL form new junctions at the distal end of the membrane protrusion.

a: A (I)Time-lapse video (video s4) of VE-cad-Venus imaged at rate of 1stack/12s during distal junction formation. New distal junctions emerge in clusters at the distal end of the JBL. Ve-cad is diffusely localized in early JBL, while it accumulates in big foci at later time points. scale bar: 2 μm. (II) Three-level thresholding of a grayscale image. The original grayscale image (I) is segmented into three intensity levels: background (white), intermediate signal (green), and strong signal (blue). (III) Tracking of VE-cad aggregates from 12s to 24s. Particle trajectories are shown as arrows from 12 s to 24 s, with arrowheads indicating the direction of movement. Dashed circles highlight dynamic aggregation of VE-cad at distal junctions. White arrowheads point distal the junction. Yellow dashed line highlights the proximal junction. b: Still-images of time-lapse video showing mRuby2-UCHD and VE-cad-Venus during JBL (0s) and distal junction formation (30s). (video s5). The dashed line encircles the protrusion. White arrow heads point distal junction foci at the distal tip of the protrusion. scale bar: 2 μm. Similar observations were made in 15 videos. c: Still images of time-lapse video showing EGFP-ZO1 and mRuby2-UCHD during the presence of proximodistal junction. After distal junction formation, F-actin gradually diminishes from proximal junction and interjunctional space, while maintaining strong localization at the distal junction. Similar observations were made in 5 videos. White arrowheads confine UCHD expression domain. scale bar: 2 μM. Yellow and white arrowheads highlight proximal and distal junction respectively. d,e: Still image of EGFP-ZO1 and mRuby2- UCHD (D), and mRuby2-UCHD and VE-cad-Venus (E) respectively, in proximodistal junction. (video s6) While junctional proteins localize more strongly at the proximal junction, respect to the new, immature distal junction, F-actin faintly localizes at the proximal junction and strongly at the distal. Similar observations were made in 10+11 videos. Yellow and white arrowheads highlight proximal and distal junction respectively scale bar: 2 μm. f: Schematic representation the spatiotemporal distribution of VE-cad, F-actin and ZO1 during formation of the distal junction in top and side view: Initially VE-cad appears diffusely dispersed throughout the membrane protrusion. Distal junction foci form at the distal side of the lamellipodia. Actin network along the distal junction. Finally, the junctional actin network along the proximal junction dissappears, as depicted by orange arrow heads.

Arp2/3 localization oscillates at the distal end of JBL.

a: Time-lapse video (video s8) of Arpc1b-Venus and ZO1-tdTomato at about 30hpf, showing deposition of Arp2/3 at the distal side of the junctional ring during 2 JBL cycles. scale bars: 2 μm. b: Time-lapse video (video s9) of Arpc1b-Venus and mRuby2-UCHD during a JBL event. scale bar: 2 μm. c and c’: Plots showing the average intensity of Arpc1b-Venus and mRuby2-UCHD along rectangular ROIs drawn along the JBL at 30 and 60 seconds, respectively. scale bar: 2 μm d: Schematic model of top and side view of spatiotemporal localization of ARP2/3, F-actin and ZO1 during JBL formation and extension for 1.5 JBL cycle.

Arp2/3 activity is required for junctional elongation and JBL formation.

a: Still-images from time-lapse videos (videos s10, s11) of JBL labelled with EGFP-UCHD, at around 32hpf, in the presence of DMSO (1%) or 1h incubation with CK666 (200μM). Similar observations were made in 10 videos. scale bars: 5 μm. Similar observations were made in more than 12 videos. b: Quantification of number of filopodia/JBL in DMSO (1%), n=10 JBL and CK666 (200μM), n=10 JBL events. Unpaired t-test was used for statistical analysis. (p-value < 0.0001). c: Quantification JBL filopodia length in DMSO (1 %), n=15 JBL and CK666 (200 μM), n=34 JBL events. d: Still images from time-lapse videos (videos s14, s15) of a ZO1-tdTomato labeled junctional rings in the presence of DMSO (1%), CK666 (200μM). Top panels t =0 and bottom panels after 1h incubation. scale bars: 10μm. e: Quantification of the junctional elongation velocity in DMSO (1%), n=21 junctions (10) embryos) and CK666 (200 μM), n=24 (10 embryos). Dotted line indicated no movement observed, black lines are medians. Unpaired t-test was used for statistical analysis. (p-value = 0.0047).

MLC is enriched at the junction poles.

a: Stills from time-lapse video (video s16) of VE-cad-Venus during junctional merging. Blue and red arrowheads point to the proximal and distal junction, respectively. The two junctions are gradually moving closer until they merge. Similar observations were made in 13 videos. scale bars 2 μm. b: Confocal image of ISV and DLAV of Myl9b-mCherry and ZO1-EGFP at around 32 hpf. Yellow arrowheads point to JBL. Dashed lines underline the distal expression domain of Myl9b-mCherry at the junctional pole. scale bars: 2 μm. c: Confocal image of DLAV and ISV immunofluorescence against GFP (green), VE-cad (magenta) and ZO1 (cyan) of an embryo expressing Myl9a-GFP at around 32hpf in a case of mosaic expression: the top cell has stronger Myl9a-GFP expression than the bottom cell (as in the schematic). c’, c’’, c’’’, c’’’’ dashed squares delimitate JBL regions of the two cells. d: quantification of Myl9-EGFP average intensity the yellow dashed squares ROIs (I) and (II) drawn on the JBL domain. The error bars indicate standard deviation between pixel intensities within the square (area of interest). e: Still images of a time-lapse video s17 showing Myl9a-GFP and mRuby2-UCHD localization during JBL formation during anastomosis of the PCeV at around 60 hpf imaged with spinning disc. White dashed lines delineate the JBL region. Yellow and white arrowheads point to the proximal and distal junction, respectively. Myl9-GFP is enriched inside the lamellipodia and localizes at the distal junction at later time points. scale bars: 2 μm.

Myosin light-chain dynamics correlates with junctional merging.

a: Still pictures of time-lapse video (video s18) of VE-cad-EGFP (Cdh5ΔC-EGFP) and Myl9b-mCherry (shown in “fire”-LUT) during junctional merging. Blue dashed lines confine the applied mask. Yellow and white arrowheads point to the proximal and distal junction, respectively. Blue and black arrowheads point to MLC accumulation at proximal and distal junction. Similar observations were made in six videos. Gray arrowheads point to newly recruited inter-junctional Myl9-mCherry. Similar observations were made in nine videos. b: Dashed blue lines delimitate 3 regions: proximal junction region (P), distal junction region (D) and inter-junctional space (I). c: Plot of average intensity of Myl9b-mCherry in the three regions over time. Signal intensities have been corrected for background levels, which has been evaluated as average of average intensities in five rectangles in the cytoplasm. scale bars: 2 μm.

Actomyosin contractility drives junctional conversion.

a: Still images from time-lapse videos (videos s23 and s24) EGFP-UCHD labelled junctional rings around 32 hpf, in the presence of DMSO (1%) or Y-27632 (45 μM). Top panels t = 0 and bottom panels 60 min incubation. scale bar: 10μm. b: Quantification of the junctional elongation velocity in DMSO (1%), n =11 junctions and Y-27632 (45 μM), n = 8. Dotted line indicated no movement observed, black lines are medians. Unpaired t-test was used for statistical analysis. p-value = 0.0081. c: Stills images from timelapse videos (videos s25 and s26) VE-cad-Venus labelled proximodistal junction, during junctional merging in the presence of DMSO (1%) or Y-27632 (75 μM). White and yellow arrowheads are pointing distal and proximal junctions respectively. scale bar: 2μm. d: Tracking of proximal-distal junction distance over time of individual junctional merging events in DMSO (1%) (green lines), and Y-27632 (75 μM) treated embryos (magenta lines). e: Quantification of the persistence of proximal and distal junction in DMSO (1%), n=13 junctional merging events and Y27632 (75 μM) n=20. Unpaired t-test was used for statistical analysis. p-value = 0.0027.

Schematic representation of the molecular mechanism of junction elongation by junction-based lamellipodia (JBL).

JBL formation is initiated by Arp2/3 activation. The JBL is pushed forward by F-actin polymerization. At the distal end a new cell-cell junction is formed. MyosinII is recruited to the interjunctional space. Actomyosin contraction merges the proximal and distal junctions resulting in an overall elongation of the junctional ring. See video s27.

The distal junction forms de novo.

a: Schematic representation of the experimental design. VE-cad-mClav2 is photoconverted at one pole of the junctional ring. Then time lapse imaging is performed on the half-converted ring. b: Time-lapse images (video s7) showing a DLAV junctional ring of an embryo expressing VE-cad-mClav2 before and right-after photoconversion. The red dashed square delimitates the photoconverted area. The white dashed square demarcates the zoomed-in area of c. c: Time-lapse of the zoomed in junctional ring pole. right-after photoconversion and 4 min later. White and yellow arrowheads point distal and proximal junction respectively. Distal junction is labeled by green, non-photoconverted VE-cad; but not by red photoconverted VE-cad. scale bars: 5μm.

CK666 disrupts Arp2/3 localization in DLAV anastomotic junctions.

Stills of Arpc1b-Venus and ZO1-td tomato before (a, c) and after 30 min treatment with DMSO 1 % (c) (n=17) and CK666 200 μM (d) (n=25). Timepoint 0 is around 30 hpf n>20. White arrow heads are pointing at junctional poles. a’, b’, c’, d’ are magnifications of the white dashed square delimited areas. scale bar: 10μm.

CK666 mediated JBL inhibition is reversible upon washout.

a: Still images from time-lapse videos (videos s12 and s13) showing F-actin dynamics at the same junctional ring, labeled with EGFP-UCHD, at around 30hpf (left) and 32hpf (right), after 30 min incubation with CK666 (200μM), and after a 90 min washout, respectively. scalebar 2μm. Similar observation were made in 11 movies. Blue and red arrowheads point to ectopic filopodia under CK666 inhibition and and reemerging lamellipodia during washout, respectively. b: Quantification of number of filopodia/JBL in CK666 (200 μM), n=11 JBL and washout n=17 JBL events. Unpaired t-test was used for statistical analysis. (P value = 0.0003). C: Quantification JBL filopodia length in CK666 (200 μM), n=11 JBL and washout n=17 JBL events. Unpaired t-test was used for statistical analysis. (p-value < 0.0001).

Myosin light-chain recruitment at the inter-junctional space during merging.

a, a’: Time-lapse of VEcad-EGFP (Cdh5ΔC-EGFP) and Myl9b-mCherry (shown in “fire” LUT) during “double junction” state (video s17). a: Still-image showing polarized accumulation of MLC during ‘‘double junction’’ state. a’: Still-images showing recruitment of MLC between proximal and distal junction. Yellow and blue arrowheads highlight the proximal and distal junction respectively b: Still-images from time-lapse video s18 of VE-cad-Venus and Myl9b-mCherry showing increase of MLC in the inter-junctional space prior to proximodistal junction mergence. Yellow and blue arrowheads highlight the proximal and distal junction. c, d: Time-lapses of VE-cad-EGFP (Cdh5ΔC-EGFP) and Myl9b-mCherry videos s19 and s20 showing recruitment of MLC between proximal and distal junction during ‘‘double junction’’ state. Yellow and blue arrowheads highlight the proximal and distal junction, respectively. scale bars: 2μm.