Angiogenesis in the developing spinal cord: blood vessel exclusion from neural progenitor region is mediated by VEGF and its antagonists.

Blood vessels in the central nervous system supply a considerable amount of oxygen via intricate vascular networks. We studied how the initial vasculature of the spinal cord is formed in avian (chicken and quail) embryos. Vascular formation in the spinal cord starts by the ingression of intra-neural vascular plexus (INVP) from the peri-neural vascular plexus (PNVP) that envelops the neural tube. At the ventral region of the PNVP, the INVP grows dorsally in the neural tube, and we observed that these vessels followed the defined path at the interface between the medially positioned and undifferentiated neural progenitor zone and the laterally positioned differentiated zone. When the interface between these two zones was experimentally displaced, INVP faithfully followed a newly formed interface, suggesting that the growth path of the INVP is determined by surrounding neural cells. The progenitor zone expressed mRNA of vascular endothelial growth factor-A whereas its receptor VEGFR2 and FLT-1 (VEGFR1), a decoy for VEGF, were expressed in INVP. By manipulating the neural tube with either VEGF or the soluble form of FLT-1, we found that INVP grew in a VEGF-dependent manner, where VEGF signals appear to be fine-tuned by counteractions with anti-angiogenic activities including FLT-1 and possibly semaphorins. These results suggest that the stereotypic patterning of early INVP is achieved by interactions between these vessels and their surrounding neural cells, where VEGF and its antagonists play important roles.

Transplantation of neural tissue: quail-chick chimeras.

Tissue transplantation is an important approach in developmental neurobiology to determine cell fate, to uncover inductive interactions required for tissue specification and patterning as well as to establish tissue competence and commitment. Avian species are among the favorite model systems for these approaches because of their accessibility and relatively large size. Here we describe two culture techniques used to generate quail-chick chimeras at different embryonic stages and methods to distinguish graft and donor tissue.

Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.

The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.

Neurovascular development uses VEGF-A signaling to regulate blood vessel ingression into the neural tube.

Neurovascular development requires communication between two developing organs, the neuroepithelium and embryonic blood vessels. We investigated the role of VEGF-A signaling in the embryonic crosstalk required for ingression of angiogenic vessel sprouts into the developing neural tube. As the neural tube develops, blood vessels enter at specific points medially and ventrally from the surrounding perineural vascular plexus. Localized ectopic expression of heparin-binding VEGF165 or VEGF189 from the developing avian neural tube resulted in supernumerary blood vessel ingression points and disrupted vessel patterning. By contrast, localized ectopic neural expression of non-heparin-binding VEGF121 did not produce supernumerary blood vessel ingression points, although the vessels that entered the neural tube became dysmorphogenic. Localized loss of endogenous VEGF-A signaling in the developing neural tube via ectopic expression of the VEGF inhibitor sFlt-1 locally blocked blood vessel ingression. The VEGF pathway manipulations were temporally controlled and did not dramatically affect neural tube maturation and dorsal-ventral patterning. Thus, neural-derived VEGF-A has a direct role in the spatially localized molecular crosstalk that is required for neurovascular development and vessel patterning in the developing neural tube.