Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish

Abstract

We examined the role of angiogenesis and the need for receptor signaling using chemical inhibition of the vascular endothelial growth factor receptor in the adult zebrafish tail fin. Using a small-molecule inhibitor, we were able to exert precise control over blood vessel regeneration. An angiogenic limit to tissue regeneration was determined, as avascular tissue containing skin, pigment, neuronal axons and bone precursors could regenerate up to about 1 mm. This indicates that tissues can regenerate without direct interaction with endothelial cells and at a distance from blood supply. We also investigated whether the effects of chemical inhibition could be enhanced in zebrafish vascular mutants. We found that adult zebrafish, heterozygous for a mutation in the critical receptor effector phospholipase Cgamma1, show a greater sensitivity to chemical inhibition. This study illustrates the utility of the adult zebrafish as a new model system for receptor signaling and chemical biology.

Figure 1

Zebrafish tail fin vasculature and ease of adult angiography. (a) Schematic diagram showing the location of the adult zebrafish heart for intracardiac injection and adult angiography. A fluorescent agent microinjected through the heart circulates throughout the fish and is visible in all the fins within minutes. (b,c) Enlarged views of the same tail fin region. (b) Pigment and bony structures of the fin ray in bright field. (c) Blood vessels after fluorescent angiography. (d) Magnified region in c of connecting blood vessels of the fin microvasculature. Scale bars, 1 cm (a); 500 μm (b,c); 100 μm (d).

Figure 2

Tail fin vessel regeneration is sensitive to VEGFR inhibition. Top panels, endothelial cells (EC, green) using a transgenic endothelial-eGFP line, Tg(fli1:EGFP)^y1. Middle panels, blood flow (Flow, red) using angiography in the same fish. Bottom panels, endothelial tip cells. (a,b) Adult zebrafish tail fins were clipped, then allowed to recover for 3 dpa. (c-f) Fish were treated with PTK787 as indicated. White boxes indicate areas where endothelial cells have not yet formed into perfused blood vessels. (g,h) Filopodia from the endothelial tip cells were found to extend into avascular tissue. Abbreviations for all figures: dpa, days post amputation; EC, endothelial cells; Flow indicates angiography. Orange arrow, amputation site for all figures. Scale bars, 500 μm (a-f); 50 μm (g); 10 μm (h).

Figure 3

Molecular analysis of regenerative angiogenesis. In situ antisense RNA hybridizations with various probes and stages in control (C) or PTK787 (PTK) treated fin samples as indicated. (a-g) In situ probes vegf-a, vegfr2, msxb, shh or fgfr1 were used on fin samples as indicated. Black arrows, clip sites in all panels. (f,g) Frozen sections of 1.2 dpa fins showing location of msxb and vegf-a mRNA staining. Scale bars, 500 μm (a-e), and 50 μm (f,g).

Figure 4

Caudal fin regrowth is limited by angiogenesis. (a-f) The same fins are shown in bright field (top panels) and with the corresponding endothelial-eGFP signal (bottom panels). Zebrafish tail fins were clipped, then allowed to recover normally or treated with PTK787 inhibitor as indicated. (g) Quantitative comparison of vessel and fin regeneration in control and PTK787-treated fish. Black bars, nonvascularized fin tissue; white bars, vascularized tissue. Average values are plotted for fin and vessel growth (n = 15). (h-m) Time-course analysis of fin recovery in control (top panels) or PTK787-treated endothelial-eGFP fish (bottom panels) in washout experiment. (n,o) Changes in vegf-a (n) or vegfr2 (o) mRNA levels standardized to β-actin during caudal fin regeneration in control (white bars) or washout where PTK787 was removed at 3 dpa (black bars). Gray bars, levels in unclipped fins. n, number of experimental replicates (pool of fins); N, number of fins used; ^*, significantly different from control (^**, P ≤ 0.01; ^***, P ≤ 0.001); #, significantly different from unclipped fin (#, P ≤ 0.05; ##, P ≤ 0.01; ###, P ≤ 0.001). Error bars indicate s.d. Scale bars, 500 μm.

Figure 5

Examination of tissues and cell types in the zebrafish tail fin. (a-f) Zebrafish tail fins were clipped and allowed to recover with or without PTK787 as indicated. In the presence of PTK787, regenerative angiogenesis is prevented as indicated by angiography in a,b. Monoclonal antibodies zns-5 or zn-12 were used to examine bone or nerve staining in c,d or e,f, respectively. Regeneration of both tissue types appears unaffected by PTK787 (d,f). (g) Transmission electron micrograph (TEM) of the caudal fin at mid-fin level. A central artery (artery) and a perivascular cell (pericyte) are indicated. (h) Higher magnification of tail fin vessels stained for smooth muscle actin using the monoclonal antibody B-4. An artery is labeled ‘a’ and a vein is labeled ‘v.’ (i-k) Frozen cross-section of a caudal fin at mid-fin level using endothelial-eGFP (EC) and smooth muscle actin (SMA) in the same sample. (l-m) Antibody staining for smooth muscle actin in 7 dpa or 14 dpa adult fins using monoclonal antibody B-4. Orange arrow, clip site in l; clip site is outside the image in m, due to the large amount of regrown tissue. Scale bars, 500 μm (a-f,l); 5 μm (g); 10 μm (h); 50 μm (i-k).

Figure 6

Effectiveness of chemical inhibitors on regenerative angiogenesis. Adult zebrafish tail fins were clipped, then allowed to recover for 3 dpa under control conditions or with inhibitors as indicated. SU5416 (a-c), SU11652 (d-f) or AAL993 (AAL; g-i).

Figure 7

Chemical analysis of regenerative angiogenesis in zebrafish lines. (a,b) Zebrafish tail fins from heterozygous PLCg1^y10/+ adults (PLC γ Het; b) and their age-matched wild-type siblings (PLCg1^+/+; WT Sib; a) were clipped and allowed to recover for 3 d in 100 nM PTK787 for partial inhibition. White arrow, clip site; orange line, total amount of tissue regrowth; green line, amount of vascularization in the regenerated tissue. (c) Tissue regeneration and the percentage of vascularization at 3 dpa were determined in zebrafish lines in controls (Control 3 dpa) or with PTK787 treatment (PTK787 3 dpa) as indicated. For each line, the sample numbers are the same for control or for inhibitor treatment: wild-type AB strain (n = 8); PLCg1^+/+ wild-type siblings (n = 5);PLCg1^y10/+ heterozygous adults (n = 12); homozygous grl^m145/m145 adults (n = 5). Error bars indicate s.d. (n = 7); heterozygous clo ^m39/+ adults Scale bar, 500 μm (a,b).