Rationale Aortic formation occurs via a recently defined morphogenic pathway. Hedgehog (Hh) signalling induces vascular endothelial growth factor (VEGF) expression, which in turn induces Notch signalling leading to correct aortic formation. Mutations in the human Parathyroid Hormone Receptor 1 (PTHR1), causes lethal chondrodysplasia, in which 50% of patients suffer aortic coarctation. The zebrafish is an excellent model to study vascular development in vivo. We therefore assessed whether PTHR1 is required for aortic formation, and whether it plays a role within the Hh-VEGF-Notch pathway.
Methodology and Results Morpholino antisense knockdown of PTHR1 induced a localised aortic occlusion at the level of the 17th somite in 50% of 2-day old embryos. The Hh inhibitor cyclopamine [100 μM] abolished mRNA expression of PTHrP (ligand for PTHR1). Using a novel Notch reporter transgenic line (csl:venus) we found that PTHR1 knockdown induced a localised defect in aortic Notch signalling at the site of the aortic occlusion. Treatment with a VEGF inducer (GS4012) failed to rescue the aortic defect in PTHR1 morphants. Upregulation of Notch signalling using an inducible system (heatshock:NotchICD) significantly rescued the aortic defect in PTHR1 morphants.
Conclusions PTHR1 signalling lies downstream of Hedgehog and VEGF, but upstream of Notch signalling to orchestrate correct patterning of a specific region of the aorta. This is most likely due to Hh regulation of the PTHR1 ligand PTHrP. These data may explain the aortic coarctation seen in human. PTHR1 mutations and for the first time implicate PTH signalling in vascular patterning and the Hh-VEGF-Notch pathway.