Figure 1. Zebrafish eng mutants develop AVMs.

(a) TALEN target site of zebrafish eng and isolated alleles. Endoglin domain structure predicted by zebrafish primary sequence: signal peptide (SP, red), Zona Pellucida domain (ZP, blue), transmembrane region (TM, orange), cytoplasmic region containing a serine/threonine-rich sequence (green) and a C-terminal PDZ-binding motif (yellow star). (b) Adult WT and eng^mu130 zebrafish. Scale bar is 10 mm. (c, d) Dorsal (c) and ventral (d) images of dissected brains from aged zebrafish. WTs exhibit hierarchical organization of vasculature, with large calibre vessels (arrows in inset). eng^mu130 zebrafish present with dilated tortuous vessels (arrowheads in inset) and loss of hierarchical patterning. Images are representative of 5 WT and 5 mut fish. Scale bar is 500 um (overview), 100 um (inset). (e) Schematic of fin regeneration model. (f-i) Still images from blood flow movies in 5 dpa fin regenerate and cartoon depiction of blood flow (arrows) in WTs (f, g) and eng^mu130 mutants (h, i). Numbers label individual rays in the movie. Arrows indicate flow direction, arrowheads highlight reversals. Numbers in parentheses depict number of rays in analysed fish sharing a similar flow characteristic (89 rays from 12 WT, and 86 rays from 12 mut). X indicates large inactive vessel. Note bleedings at distal tips of regenerating rays in eng^mu130 fish. Scale bar is 200 um. (j, k) Maximum intensity projections of confocal z-stacks of AVM in eng^mu130 regenerate and comparable region in WT at 5 dpa. Dashed line indicates amputation plane. Solid lines indicate vessel calibre and yellow bracket indicates region of vasculature analysed (inset). Cartoon depicts blood flow patterns in movies. Note shunting of arterial blood to the vein in mutant AVM, and flow reversal in distal part of affected vein (arrowhead). Scale bar is 400 um (overview), 50 um (inset). (l-p) Quantification of diameter (l), endothelial cell (EC) number (m), endothelial area (n) and cell density (o) in arteries and veins in proximity to AVM in eng^mu130 fish and comparable WT regions (n=9 WT, n=9 mut). Analysed by paired Student’s t-test.

n.s., not significant, *P<0.05, **P<0.01, ***P<0.001, error bars indicate s.e.m.