Intersegmental vessel formation in zebrafish: requirement for VEGF but not BMP signalling revealed by selective and non-selective BMP antagonists

Abstract

Background and purpose: Bone morphogenetic proteins (BMPs) were first identified through their role in inducing bone and cartilage formation, but many other important functions have since been ascribed to BMPs, including dorsoventral patterning, angiogenesis and tissue homeostasis. Using dorsomorphin and LDN193189, selective small molecule inhibitors of BMP signalling, we investigated the role of BMP signalling in early vascular patterning in zebrafish.

Experimental approach: The effects of dorsomorphin and LDN193189 on vascular endothelial growth factor-a (VEGF) and BMP signalling in developing zebrafish and in human pulmonary artery endothelial cells were determined using confocal microscopy, Western blotting and quantitative PCR.

Key results: We showed that dorsomorphin, similar to the VEGF inhibitor SU5416, strongly inhibits intersegmental vessel formation in zebrafish and that this is due to inhibition of VEGF activation of VEGF receptor 2 (VEGFR2), leading to reduced VEGF-induced phospho-ERK (extracellular regulated kinase) 1/2 and VEGF target gene transcription. These effects occurred at concentrations of dorsomorphin that block BMP signalling. We also showed that LDN193189, an analogue of dorsomorphin, more potently blocks BMP signalling but has no effect on VEGF signalling in zebrafish and does not disrupt early vascular patterning.

Conclusions and implications: Dorsomorphin inhibits both BMP and VEGF signalling, whereas LDN193189 is a more selective BMP antagonist. Results obtained in cardiovascular studies using dorsomorphin need to be interpreted with caution, and use of LDN193189 would be preferable due to its selectivity. Our data also suggest that BMP signalling is dispensable for early patterning of intersegmental vessels in zebrafish.

Figure 1

Dorsomorphin dorsalized zebrafish embryos. (A) Normal and classes of dorsalized embryos at 24 hpf (hours post fertilization) (unless stated). Lateral views with anterior to left. Class 1 only has loss of distal ventral fin, and class 2 has complete loss of ventral fin. Black arrows highlight loss of ventral fin tissue. Scale bars indicate 500 µm. (B) Summary of dorsalization class when embryos were exposed to different concentrations of dorsomorphin (DM) or 10 µM dimethylsulphoxide (DMSO) vehicle control from 4 hpf. Graph illustrates the combined results of class scoring from both 11 and 24 hpf; n= 121–143 embryos per group.

Figure 2

Dorsomorphin blocked intersegmental vessel (ISV) formation. (A) Normal embryo at 30 hpf (hours post fertilization) with red box illustrating the location where the confocal images were taken. Representative confocal z-stack images of Tg(fli1a:egfp)^y1 zebrafish. (B) Normal with dorsal aorta (white bracket), posterior cardinal vein (yellow bracket) and ISV (white arrow) highlighted. Zebrafish exposed to 10 µM dorsomorphin (C) or 1 µM SU5416 (a pan vascular endothelial growth factor-a receptor inhibitor) (D) from 12 hpf have loss of the dorsal aorta and ISVs and expansion of the posterior cardinal vein. All are lateral views with anterior to left. Scale bars indicate 200 µm; n= 60–80 each group.

Figure 3

Dorsomorphin inhibited signalling pathways induced by vascular endothelial growth factor-a (VEGF) and bone morphogenetic protein (BMP)6. Human pulmonary artery endothelial cells were pretreated with 0.1% fetal bovine serum (FBS), 10 µM dimethylsulphoxide (DMSO) (vehicle control), 2.5 or 10 µM dorsomorphin (DM) and then with 0.1% FBS or 25 ng·mL⁻¹ VEGF (A, B) or 0.1% FBS or 50 ng·mL⁻¹ BMP6 (C, D). VEGF induced phosphorylation of ERK 1/2 (A), VEGF target genes Nor1 and Nur77 (B) when cells were pretreated with 0.1% FBS and 10 µM DMSO, but this was completely inhibited by both concentrations of dorsomorphin. BMP6 induced phosphorylation of Smad 1/5 (C) and Id1 (D) when cells were pretreated with 0.1% FBS and 10 µM DMSO but again this was completely inhibited when cells were treated with dorsomorphin. VEGF induced phosphorylation of VEGF receptor 2 (VEGFR2) at tyrosine 1175 but this was blocked by 10 µM dorsomorphin (E). A, C and E are representative blots and B and D show mean ± SD, all n= 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with 0.1%/0.1% serum control.

Figure 4

LDN193189 dorsalized zebrafish embryos but had no effect on vascular development. (A) Summary of combined dorsalization class at 11 and 24 hpf (hours post fertilization) when embryos were exposed to different concentrations of LDN193189 1 µM or dimethylsulphoxide (DMSO) vehicle control from 4 hpf; n= 130–153 embryos per group. (B) Representative confocal z-stack image (location as per red box in Figure 2A) of a Tg(fli1a:egfp)^y1 zebrafish embryo taken as per Figure 2A exposed to 1 µM LDN193189 from 12 hpf with normal blood vessel development at 30 hpf. Lateral view with anterior to left. Scale bar indicates 200 µm; n= 60.

Figure 5

LDN193189 inhibited signalling pathways induced by bone morphogenetic protein (BMP)6 but not those induced by vascular endothelial growth factor-a (VEGF). Human pulmonary artery endothelial cells were pretreated with 0.1% fetal bovine serum (FBS), 1 µM dimethylsulphoxide (DMSO) (vehicle control) or 1 µM LDN193189 (LDN) and then with 0.1% FBS or 25 ng·mL⁻¹ VEGF (A, B) or 0.1% FBS or 50 ng·mL⁻¹ BMP6 (C, D). VEGF induced phosphorylation of extracellular regulated kinase (ERK) 1/2 (A), VEGF target genes Nor1 and Nur77 (B) when cells were pretreated with 0.1% FBS, 1 µM DMSO and 1 µM LDN193189. BMP6 induced phosphorylation of Smad 1/5 (C) and Id1 (D) when cells were pretreated with 0.1% FBS and 1 µM DMSO, but this was completely inhibited when cells were treated with LDN193189. A and C are representative blots and B and D show mean ± SD, all n= 3. *P < 0.05, **P < 0.01, compared with 0.1%/0.1% serum control.