Vascularization of the embryonic spinal cord is crucial for CNS development and homeostasis. In early embryogenesis, there are no endothelial cells (ECs) or endothelial cell precursors (angioblasts) in the neuroectoderm, nor can cells within this tissue give rise to ECs [1
]. Almost as soon as the anlagen CNS forms a tube, it begins communicating with the surrounding mesodermal tissue—where angioblasts and endothelial cells reside. In this narrow developmental window (E8.5–E10.0 in mouse or Day2–4 in avian embryos), the neural tube recruits angioblasts and ECs to coalesce into a ring of vessels, known as the peri-neural vessel plexus (PNVP) (). This is the first blood vessel patterning process coordinated by the neural tube of the CNS. As neural development proceeds within the neural tube, blood vessels invade the neuroectoderm via sprouting angiogenesis—forming an intra-neural vessel plexus (INVP) (). This is the second major blood vessel patterning event coordinated by the CNS. Both the PNVP and INVP acquire specific properties unique to CNS vasculature that form the blood brain barrier (BBB) [5
2.1 Formation of the peri-neural vessel plexus (PNVP)
The neural tube was identified as the source tissue for positive blood vessel patterning signals capable of inducing EC migration and directing PNVP formation. Ectopically grafted, mouse-derived neural tubes in avian hosts recruited a PNVP, whereas grafted acrylic beads, or notochords (an embryonic midline patterning structure) did not [9
]. Furthermore, analysis of Tbx6 mutant mouse embryos, with defects in paraxial mesoderm specification resulting in the formation of multiple neural tubes lateral to the endogenous neural tube, revealed that these ectopic neural tubes also have the ability to recruit PNVPs [9
]. These experiments clearly demonstrate that the neural tube is the source of a diffusible blood vessel patterning signal coordinating PNVP formation. Furthermore, the ability of the neural tube to pattern a PNVP is not context dependent, as ectopic neural tubes induced patterning of ECs in multiple vascular beds.
2.2 Role of neural tube-derived VEGF-A in PNVP formation
A major component of the neural tube-derived signal is Vascular Endothelial Growth Factor-A (VEGF-A) (). VEGF-A is the most ubiquitous and potent angiogenic factor known, and has well-established pro-angiogenic effects on ECs and blood vessel patterning during development [11
]. VEGF-A expression analysis in a LacZ reporter strain showed that VEGF-A was up-regulated in the neural tube just prior to PNVP recruitment and its expression became localized to the outer, pial surface of the neural tube as the PNVP developed [9
]. Explanted mouse pre-somitic mesodermal tissue, a known source of angioblasts and ECs that colonize the PNVP in vivo
, formed a blood vessel plexus when cultured in a collagen matrix either with VEGF-A protein or avian neural tubes; however, this plexus did not form when PSM is cultured with neural tubes in the presence of VEGF-A inhibitors [9
]. This experiment demonstrated that the neural tube stimulates blood vessel plexus formation, and this effect is dependent on VEGF-A signaling.
VEGF-A modulates blood vessel patterning in several major ways. Proper VEGF-A expression levels in the developing embryo are required for embryonic survival. Inactivation of one vegf-a
allele resulted in embryonic lethality due to cardiovascular defects in mice between E11–12 [12
]. Conversely, global increases in VEGF-A expression, produced by insertion of a modified vegf-a
gene into the endogenous locus, resulted in embryonic lethality due to cardiovascular defects and vessel overgrowth at E12.5 [14
]. Reduction of CNS-derived VEGF-A in mice resulted in early postnatal lethality, reduced blood vessel density in the brain, increased neuronal apoptosis, and degeneration of the cerebral cortex [15
]. These studies did not analyze effects on PNVP formation; however, there is evidence to suggest that altered CNS-derived VEGF-A levels can perturb PNVP patterning. Avian neural tubes electroporated with human vegf-a
cDNA in a gain of function experiment, exhibited an increase in PNVP vessel thickness [17
]. Neural tubes electroporated with high amounts of sflt1
transgene, a soluble flt1
(also known as vegfr1
) to trap VEGF-A, resulted in missing sections of the PNVP or thinner PNVP vessels in some instances (J. M. James, unpublished data).
VEGF-A can also act both as a long-range and short-range vessel patterning cue to locally guide vessel sprouts [18
]. It achieves this signaling range because it is alternatively spliced into at least 6 isoforms, three of which are abundantly expressed in the mouse CNS: VEGF120
, and VEGF188
]. Each isoform interacts differently with the extracellular matrix via heparin binding sites [22
has two heparin-binding sites and is the least soluble, providing short-range or precise vessel patterning, while VEGF120
has no matrix-binding sites, is diffusible, and can provide long-range vessel pattering signals. VEGF165
has one heparin-binding site and maintains intermediate properties [23
]. Mice expressing only one of the three major isoforms had intraneural vessel patterning defects (refer to section 2.4)
; however, PNVP formation around the spinal cord and hindbrain regions of the neural tube appeared normal in these mutants (J. M. James, unpublished data, [19
]). Taken together, the level of neural tube-derived VEGF-A is important for the PNVP formation whereas matrix-binding properties of VEGF-A may not be required for the recruitment of ECs and angioblasts to form the PNVP.
2.3 Formation of the intra-neural vessel plexus (INVP)
The PNVP begins forming approximately one day before angiogenic sprouts invade the neural tube. Vessel sprouts entering the neural tube do so in highly stereotypical locations () [2
]. In the developing chick embryo, sixteen distinct blood vessels form reproducibly along the dorsal-ventral axis (transverse plane) of the neural tube before the neural tube becomes uniformly vascularized. Each vessel forms along both spatial and temporal axes, and earlier vessels act as a scaffold onto which subsequent vessels can anastomose with or sprout from [24
]. These patterning events take place at regular intervals along the length of the chick neural tube. The discovery that a hierarchal system of intraneural blood vessel growth and patterning exists, suggests that neural tube angiogenesis is highly regulated by neural tube-derived signals.
Many signaling molecules with known roles in vascular patterning are expressed in the neural tube. Perhaps it is best to think of signals regulating INVP development as layers of complexity. In this section, we first discuss how neural tube-derived VEGF-A is important for blood vessel ingression, proper vascular density, and fine–tuning of the blood vessel pattern. Second, recent evidence shows that Wnt subfamily members promote acquisition of BBB characteristics in intra-neural vessels. Lastly, neural cells associate with blood vessels to develop neuro-vascular units, which function to pattern and stabilize the INVP, as well as maintain the integrity of the BBB. Each signaling layer builds on the one before it, giving us an overall picture of how a reproducible blood vessel pattern is achieved and maintained within the neural tube.
2.4 Role of neural tube-derived VEGF-A in INVP formation
In the avian spinal cord, inhibition of VEGF-A-signaling via electroporation of a soluble flt1
resulted in an almost complete lack of vessel invasion [17
]. Sequestering VEGF-A or inhibiting VEGF-A signaling in vitro
and in vivo
also profoundly disrupted INVP formation in dorsal root ganglia (DRG) of the PNS, indicating that VEGF-A signaling is crucial for INVP formation in both the CNS and PNS [25
]. Genetic studies in mice that express a single VEGF-A isoform provided evidence that VEGF-A isoform expression influences the INVP pattern. Vegf120/120
neural tubes displayed delayed ingression and reduced sprout number. Vegf188/188
neural tubes had hyper-branched, thin vessels, while vegf165/165
mutants had phenotypically normal blood vessels (J. M. James, unpublished data). These observations are consistent with reports describing vessel branching and morphogenesis defects within the hindbrain as well as globally in the isoform mutant mice [19
]. In avian neural tubes, over-expression of matrix-binding VEGF-A resulted in supernumerary and ectopic vessel sprout formation, whereas over-expression of soluble, non-matrix-binding VEGF-A did not [17
]. These results suggest that matrix-binding VEGF-A can give precise patterning information to the blood vessels, directing vessel ingression patterns, and that matrix-binding VEGF-A partially directs the exact timing of blood vessel invasion into the neural tube.
Neuropilin-1 (NRP1) is also important for mediating isoform-specific VEGF-A signaling. NRP1, a co-receptor for VEGF-A, is expressed in ECs and has been shown to enhance VEGF-Flk1 interactions in vitro
by forming a receptor complex with Flk1 (also known as VEGFR2) [27
]. NRP1 is also highly expressed on neuronal axons and acts as a co-receptor for Semaphorin molecules, forming a complex with Plexin receptors [28
]. Although NRP1 can bind all three major VEGF-A isoforms, VEGF120
is too small to bridge the gap between Flk1 and NRP1 [29
], thus precluding complex formation. The lack of the Flk1-NRP1-VEGF complex is thought to partially account for the severity of blood vessel defects in the vegf120/120
mice. Conventional nrp1
mutant mice displayed normal blood vessel ingression and blood vessel density within the embryonic hindbrain, however there were defects in lateral blood vessel branching as vessel sprouts interface the ventricular zone [30
]. Endothelial-specific deletion of nrp1
had a slightly different effect, resulting in the formation of large, un-branched vessels within the neural tube [31
], similar to the vessel phenotype in vegf120/120
mutants. Taken together, VEGF-A/NRP1 signaling in ECs is important for proper INVP formation in the CNS.
Though much is known about the importance of VEGF-A signaling in proper development of the CNS vasculature, we still do not know if matrix-binding VEGF-As are localized to precise points of blood vessel ingression and branching, thus directing intricate vessel patterning (, model #1). Another possible mechanism to explain stereotypic blood vessel ingression is that VEGF-A-mediated positive spatial signals are balanced by negative spatial signals that prevent blood vessel ingression from the PNVP (, model #2). Since ectopic expression of sFlt-1 inhibited INVP formation in the neural tube [17
] and vessel-derived sFlt-1 was required for local sprout guidance [32
], local sFlt1 may neutralize VEGF-A secreted from the neural tube to inhibit vessel ingression.
Recent studies demonstrated that molecules regulating axonal guidance and patterning also regulate blood vessel patterning, and the tip cell of a growing vessel acts much like an axonal growth cone [33
]. Repulsive axon guidance molecules may coordinate with VEGF-A signaling to pattern blood vessel ingression in the neural tube. Netrin signaling through UNC5 receptor homodimers or UN C5-DCC receptor heterodimers provides repulsive cues to ECs [35
]. Netrins inhibited VEGF-A-induced EC migration in vitro
and VEGF-A-driven angiogenesis in vivo.
Semaphorin 3 (Sema3) subfamily members are expressed in the neural tube and also act as repulsive cues in axon guidance and neuronal cell migration [37
], as well as EC migration. Sema3s signal through Plexin/NRP1 receptor complexes expressed on axons and ECs. Given that NRP1 acts as a VEGF-A and a Sema3 co-receptor, the inhibitory effect of Sema3s on angiogenic sprouting and EC migration was attributed to Sema3/VEGF-A competition for NRP1 binding [38
]. However, recent biochemical studies demonstrated that Sema3A and VEGF-A bind to distinct domains of NRP1 [41
] and induce NRP1 endocytosis through different pathways, suggesting that these NRP1 ligands function separately, rather than competitively. An alternative molecular mechanism for explaining the effects of Sema3s on vascular development is suggested by the endothelial expression of plxnd1
in both mouse and zebrafish embryos. Paracrine Sema3 (Sema3E in mouse) signals from the somites blocked ingression of plexinD1 expressing ECs from the dorsal aorta via a repulsive mechanism, resulting in the patterned segmental vasculature between somite boundaries [43
]. An intriguing idea is that these repulsive guidance signals in combination with a VEGF-A-mediated positive guidance signal control the precise vessel ingression pattern in the neural tube (, model #2). However, the requirement for axon guidance signals in neuronal development makes it difficult to distinguish whether defective INVP formation and patterning in mutants lacking these guidance genes is due to the genetic perturbation of the guidance cues to ECs or to secondary effects from a disruption of neuronal patterning. To circumvent this problem, EC-specific deletion of cell-autonomous components of these signaling pathways is required.
2.5 Role of neural tube-derived Wnts in INVP formation
The canonical Wnt signaling pathway regulates crucial aspects of cardiovascular development [44
]. Wnt7a and Wnt7b are highly expressed by the embryonic spinal cord, whereas canonical Wnt signaling is active in both PNVP and INVP ECs. The CNS-specific deletion of wnt7a
as well as EC-specific deletion of β-catenin
in mice, resulted in severe CNS-specific hemorrhage due to dilated PNVP and defective INVP ECs and pericytes in the neural tube [45
]. These results indicated that canonical Wnt signaling in ECs is important for PNVP integrity and blood vessel ingression to form the INVP ().
CNS-specific hemorrhage observed upon CNS ablation of wnt7a
as well as EC-specific ablation of β-catenin
highlights the role of canonical Wnt signaling in BBB development. The BBB prevents movement of molecules and ions between blood circulation and the CNS. CNS ECs are joined at their apical surface by a belt-like ring of tight junctions to prevent para-cellular transport. Transplantation studies suggested that BBB formation is not intrinsic to CNS ECs. Instead, the CNS directly induces the BBB characteristics in ECs by interaction with the CNS cells such as neural progenitors during early developmental stages [47
]. Wnt7 subfamily signaling is sufficient to induce BBB characteristics in ECs based on Wnt7a induction of Glut1 (a glucose transporter) and other BBB-specific influx transporters in cultured ECs and ectopic Wnt7a induction of Glut1 in non-CNS vasculature in mice carrying a wnt7a
] (). As expected, both neuronal wnt7a/7b
mutants and endothelial β-catenin
mutants had reduced expression of the BBB-specific influx transporter in the PNVP and INVP [46
An important question for future studies is how canonical Wnt signaling interacts with other signaling pathways like the VEGF-A pathway to control PNVP and INVP formation in the neural tube. Canonical Wnt signaling appears to have dual functions in patterning the CNS vasculature: one in regulating blood vessel ingression and also a role in BBB development. The gain-of-function
manipulation of wnt7a
induced a profound effect on Glut1 induction in non-CNS vasculature, but did not display ectopic or enhanced vessel ingression, although the loss of function manipulation of both wnt7a
had severe defects in vessel ingression [46
]. These observations raise an intriguing possibility that Wnt7s may be permissive signals, required for VEGF-A-mediated vessel ingression to proceed normally, whereas Wnt signaling is instructive for acquisition of BBB characteristics in ECs.
2.6 Establishment of neuro-vascular units for BBB development
ECs forming the BBB require interactions with other cell types in the CNS parenchyma such as pericytes (PCs), neurons, and astrocytes in order to maintain blood vessel integrity and BBB characteristics (). Collectively, these cell complexes are called neuro-vascular units. Disruption of these units leads to mis-pattering of the CNS blood vessels and severe intra-neural hemorrhage. One major mechanism regulating formation of functional neuro-vascular units is integrin-mediated, TGFβ signaling. Three TGFβ isoforms (TGFβ1, TGFβ2, TGFβ3) are secreted from cells in an inactive form, associating with the Latent Associated Peptide (LAP). Integrins are transmembrane cell-adhesion proteins, composed of an alpha (α) and beta (β) subunit, that are required to cleave the LAP from latent TGFβ. Within the context of the developing CNS vasculature, TGFβ1 and TGFβ3 activation by αv/β8 integrin is required for proper neuro-vascular development. Both TGFβ and αv/β8 integrin are expressed by radial glial cells as ECs begin to invade the neural tube. Mice carrying mutations in both tgfβ1
genes developed cerebral hemorrhages from E11.5 and subsequent embryonic lethality [48
]. Interestingly, mice with mutations in αv
or β8 integrin
also developed cerebral hemorrhages [50
]. These studies strengthen the evidence supporting a critical role for TGFβ signaling in neuro-vascular unit formation; however, which components in the neuro-vascular unit are influenced by TGFβ signaling remains unclear.
PCs are recruited to the endothelium during vascular development. Evidence from in vitro
experiments as well as genetic studies in mice has suggested that while TGFβ signaling appears to be critical for early PC differentiation, platelet-derived growth factor (PDGF)/PDGF receptor-β (PDGFR-β) signaling seems to be involved in later proliferation and migration of these cells (). Because the EC-specific pdgfb
mutant exhibited reduced PC recruitment and brain microhemorrhages [52
], this suggests that proper PC recruitment is important for the formation of the neuro-vascular unit. Recent exciting studies suggest that PC recruitment to CNS vasculature, prior to astrocyte development, is necessary for the BBB formation [53
]. PCs strengthen EC barrier formation through regulation of tight junction structure during embryogenesis (). While much is known about the cellular dynamics of PC recruitment and function in the neuro-vascular unit, the origin and specification of PCs in the CNS vasculature remain elusive.
Here we propose one intriguing model to explain CNS vascular development: ECs are recruited by neural tube-derived VEGF-A to form the PNVP (). PNVP ECs are induced to express BBB specific genes, such as Glut1, by interaction with CNS cells such as neural progenitors. This process seems to be largely regulated by canonical Wnt signaling (). Further, functional EC integrity to form the BBB is regulated by PCs and subsequently other components of the neuro-vascular unit such as astrocytes (). Stereotypic patterning of the INVP is regulated by a combination of positive spatial cues (VEGF-A and Wnt7a/7b) and unidentified negative spatial cues ().
Communication between blood vessels and CNS tissues (e.g. neural tube) during early embryonic development is crucial for the formation of CNS vasculature. The potential for therapeutic manipulation in instances of BBB impairment such as multiple sclerosis (MS), hereditary hemorrhagic telangiectasia (HHT), and brain tumors, underscores the need to better understand what signaling pathways regulate the CNS vasculature during development.