Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish

Abstract

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1(+) HSCs.

Figure 1. Zebrafish ptc1;ptc2 double mutants fail to establish circulation and exhibit multiple endothelial defects and defective angioblast migration

A, B) ptc1;ptc2 mutant (B) embryos at 27hpf showed somitic flattening (white arrowhead) and loss of lens (asterisk). ptc1;ptc2 embryos showed non-circulating primitive erythrocytes in the posterior ICM (red arrowhead). C, D) cdh5 expression in ptc1;ptc2 mutants (D) revealed defective formation of the CCV/DC (black arrowheads) and PCV (white arrowheads) compared to wild-type (C). E, F) Confocal image of trunk vasculature in Tg(fli1a:EGFP)y1;Tg(gata1:dsred)sd2/+ (E) and Tg(gata1:dsred)sd2/+;ptc1;ptc2 (F) embryos, anterior to the left, posterior right. The primary vasculature formed normally in Tg(fli1a:EGFP)y1;Tg(gata1:dsred)sd2/+ embryos, and circulation commenced normally (yellow arrowhead and Movie S1), whilst Tg(fli1a:EGFP)y1;Tg(gata1:dsred)sd2/+;ptc1;ptc2 embryos had disorganised vasculature, axial vessels were non-continuous along their A-P axis (asterisks) and embryos lacked circulation (yellow arrowhead and Movie S2). G-L) Confocal images using a Tg(fli1a:EGFP)y1/+ background to visualise migrating angioblasts in indicated genetic backgrounds. Dorsal views are shown. G-I) Normal angioblast migration in Tg(fli1a:EGFP)y1/+ embryos. (J-L) Fewer angioblasts migrated to the midline in Tg(fli1a:EGFP)y1/+;ptc1;ptc2 embryos and formed a discontinuous endothelial cord by 18s (Movie S3 and white arrowheads). Angioblasts were present in more lateral positions (Movie S4 and L, yellow arrowheads) than in controls at the corresponding stage (I, yellow arrrowheads). CCV common cardinal vein, PLM posterior lateral mesoderm, ICM intermediate cell mass.

Figure 2. ptc1;ptc2 mutants exhibit precocious vessel sprouting from the posterior cardinal vein

Confocal images of developing trunk vasculature in Tg(fli1a:EGFP)y1/+ (A, C, E) and Tg(fli1a:EGFP)y1/+;ptc1;ptc2 embryos at 27hpf (B, D, F), anterior is left, posterior right. A) Normal trunk vasculature. Highlighted area is shown in panel C. B) Endothelial connections between the DA and PCV were present in Tg(fli1a:EGFP)y1/+;ptc1;ptc2 embryos (grey arrowheads) and ectopic vessel sprouts arising from PCV were present (B, D white arrowheads). Highlighted area is shown in panel d. 3D reconstruction showing transverse plane of embryo shown in panels A, C (E) and embryo shown in panels B, D (F) shows the sprouting capacity of ECs in the PCV region in ptc1;ptc2 mutants (white arrowhead).

Figure 3. ptc1; ptc2 mutants exhibit ectopic arterial differentiation at the expense of venous differentiation, resulting in conversion of the PCV into a second artery

Lateral views of trunk region are shown, oriented anterior to left, posterior right. A) kdrl exhibited arterial preference in wild-type embryos (red arrowheads), but was present ectopically in the PCV in ptc1;ptc2 embryos (B, blue arrowheads). C) ephrinb2a was restricted to the DA in wild-type embryos (red arrowhead) but present ectopically in the PCV in ptc1;ptc2 embryos (blue arrowheads). E) aplnra and flt4 (G) expression was restricted to the PCV in wild-type embryos (blue arrowheads) and downregulated in ptc1;ptc2 embryos (F, H, blue arrowheads). I) runx1 was restricted to the DA in wild-type embryos (white arrowhead), downregulated in the DA of ptc1;ptc2 embryos (J, white arrowhead) and ectopically expressed in the PCV of ptc1;ptc2 embryos (J, black arrowheads). K) Increased trunk vegfa expression was present in ptc1;ptc2 embryos (L, green arrowhead) in comparison to wild-type (K, green arrowhead).

Figure 4. Somitic wnt16-deltaC/deltaD signalling required for HSC specification is abrogated in ptc1;ptc2 mutants

Zebrafish flat mounts are shown with anterior to the left and posterior to the right. A, B) somitic wnt16 expression is strongly downregulated in ptc1;ptc2 embryos at 10s (B, black arrowheads) and this downregulation persisted at 15s stage (C, D black arrowheads). E, F) Expression of deltaC was absent from the somitic mesoderm (SM) of ptc1;ptc2 embryos (F, black arrowheads), but was present within the pre-somitic mesoderm (PSM) (F, grey arrowheads). G, H) deltaD expression was strongly downregulated within the SM (H, black arrowheads) and PSM (H, grey arrowheads) of ptc1;ptc2 embryos.

Figure 5. Arterial specification occurs independently of VEGF signalling in ptc1;ptc2;plcg1 triple mutants

(A-E) Tg(fli1a:EGFP)y1/+;ptc1;ptc2 embryos formed two separate vessels, both with sprouting capacity (B), whilst Tg(fli1a:EGFP)y1/+;plcg1^t26480 embryos formed a single vessel without ISV sprouts lacking arterial gene expression (data not shown)(C). Two separate vessels were present in triple mutant embryos (D, zoomed view in E), but no ISVs formed. kdrl (F, G) and ephrinb2a (H, I) expression indicated that the DA remained duplicated in the triple mutants, albeit with fewer cells.

Figure 6. Arterial specification in ptc1;ptc2 mutants is dependent upon Notch signalling

A) Tg(fli1a:EGFP)y1/+;ptc1;ptc2 embryos exhibit blood vessel formation (B, yellow arrowhead) (n= 15/230) even following Su(H) morpholino injection (B, yellow arrowhead) (n= 5/95) C) Normal DA ephrinb2a expression in uninjected non-double mutant embryos (C, red arrowhead) (n=157/167) D) Loss of ephrinb2a in DA of non-double mutant Su(H) morphants (red arrowhead) (n=159/180) E) Increased (red arrowhead), ectopic (green arrowhead) ephrinb2a expression in uninjected ptc1;ptc2 embryos (n=10/167) F) Loss of ephrinb2a in DA of ptc1;ptc2 Su(H) morphants (n=16/180)

Figure 7. Knockdown of crlrla in combination with VEGF inhibition prevents arterial differentiation in ptc1;ptc2 mutants

A) Wild-type somitic (green arrowhead) and DA crlra expression (red arrowhead) B) Increased somitic (green arrowhead) and DA (red arrowhead) crlra expression in ptc1;ptc2 embryos. C) normal somitic crlra expression in uninjected wild type non-double mutant embryos (arrowhead) (n=58/66) D) Decreased somitic crlra expression in non-double mutant crlra morphants (n=58/71). E, F) No difference in crlra expression (arrowheads) was detected between uninjected (n=5/66) and crlra morphant (n=4/71) ptc1;ptc2 embryos. Uninjected wild-type sibs treated with SU5416 from tailbud until collection exhibited total absence of vascular ephrinb2a expression (K, red arrowhead) (n=40/50) compared to controls (G, red arrowhead) (n=46/49). SU5416 treated ptc1;ptc2 mutants showed increased ephrinb2a (L, red arrowhead) and ectopic expression in PCV region (L, blue arrowhead) (n=4/50), as did uninjected ptc1;ptc2 mutants (H, red and blue arrowheads) (n=3/49). Uninjected SU5416 treated ptc1;ptc2 embryos exhibited no vessel sprouting in comparison to DMSO treated ptc1;ptc2 embryos. ephrinb2a expression was downregulated in the DA of non-double mutant crlra morphants treated with DMSO (I, red arrowhead) (n=78/96), whilst non-double mutant crlra morphants treated with SU5416 exhibited a loss of vascular ephrinb2a expression (M, red arrowhead) (n=165/183). ptc1;ptc2 crlra morphants treated with DMSO showed strong ephrinb2a expression (J, red arrowhead), which was present ectopically in the region of the PCV (J, blue arrowhead) (n=8/96), whilst vascular ephrinb2a was absent in ptc1;ptc2 crlra morphants treated with SU5416 (N, red arrowhead) (n=10/186). O) Proposed model for arterial differentiation.