Connecting Vascular and Nervous System Development: Angiogenesis and The Blood-Brain Barrier

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Annu. Rev. Neurosci. 2010.33:379-408. Downloaded from www.annualreviews.org by Instituto Tecnologico de Costa Rica (ITCR) on 11/02/13. For personal use only.
Connecting Vascular and Nervous System Development: Angiogenesis and the Blood-Brain Barrier
Stephen J. Tam and Ryan J. Watts
Neurodegeneration Labs, Department of Neuroscience, Genentech, Inc., South San Francisco, California 94080; email: watts.ryan@gene.com
Annu. Rev. Neurosci. 2010. 33:379408 First published online as a Review in Advance on April 1, 2010 The Annual Review of Neuroscience is online at neuro.annualreviews.org This articles doi: 10.1146/annurev-neuro-060909-152829 Copyright c 2010 by Annual Reviews. All rights reserved 0147-006X/10/0721-0379$20.00
Key Words
endothelial cells, axon guidance, neurovascular unit, astrocytes, tight junctions, Wnt signaling
Abstract
The vascular and nervous systems share a common necessity of circuit formation to coordinate nutrient and information transfer, respectively. Shared developmental principles have evolved to orchestrate the formation of both the vascular and the nervous systems. This evolution is highlighted by the identication of specic guidance cues that direct both systems to their target tissues. In addition to sharing cellular and molecular signaling events during development, the vascular and nervous systems also form an intricate interface within the central nervous system called the neurovascular unit. Understanding how the neurovascular unit develops and functions, and more specically how the bloodbrain barrier within this unit is established, is of utmost importance. We explore the history, recent discoveries, and unanswered questions surrounding the relationship between the vascular and nervous systems with a focus on developmental signaling cues that guide network formation and establish the interface between these two systems.
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Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . ANATOMICAL PATTERN OF THE VASCULAR AND NERVOUS SYSTEMS . . . . . . . . . . . . Evolution of the Nervous and Vascular Systems . . . . . . . . . . . Shared Anatomical Patterning . . . . . . SHARED CELLULAR MECHANISMS . . . . . . . . . . . . . . . . . . . The Cellular Organization of the Nervous and Vascular Systems. . . The Axon Growth Cone and Endothelial Tip Cell . . . . . . . . . . . . SHARED MOLECULAR MECHANISMS . . . . . . . . . . . . . . . . . . . Axon Guidance Molecules and Angiogenesis . . . . . . . . . . . . . . . The Slits and Robos in Vascular Guidance . . . . . . . . . . . The Semaphorins/Plexins/Nrps in Vascular Guidance . . . . . . . . . . . The Netrin/Unc5/DCC Family in Vascular Guidance . . . . . . . . . . . The Ephrin/Eph Family in Vascular Guidance . . . . . . . . . . . THE INTERFACE BETWEEN THE VASCULAR AND NERVOUS SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . Molecular Properties of the Blood-Brain Barrier . . . . . . . . . . . . . The Neurovascular Unit . . . . . . . . . . . CNS Angiogenesis and BBB Formation: A Role for Wnt Signaling . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . . 380
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CNS: central nervous system NVU: neurovascular unit BBB: blood-brain barrier
INTRODUCTION
The anatomical similarities between the vascular and nervous systems have been recognized for centuries. Recent discoveries have continued to deepen this apparent relationship at both the cellular and the molecular
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levels. At a gross anatomical level, the pathways of blood vessels and nerves appear congruous. At the cellular level, extending vessels and neurons utilize similar specialized structures that drive target tissue guidance through sensing their local environment. Ultimately these similarities can be traced back to several families of shared molecular guidance cues that guide both endothelial cells and neurons (Carmeliet & Tessier-Lavigne 2005, Dickson 2002, Larriv ee et al. 2009, ODonnell et al. 2009, Tessier-Lavigne & Goodman 1996). Although still in its early years, the eld of vascular guidance has taken many cues from past insights provided by the axonal guidance principles of attraction and repulsion. Beyond the common anatomical, cellular, and molecular properties, the vascular and nervous systems have developed an intricate relationship within the central nervous system (CNS) itself. This connection is found at the interface between the vascular and nervous systems, generally known as the neurovascular unit (NVU). The NVU is composed of endothelial cells, pericytes, glia, and neurons, which are tightly coupled to control cerebrovascular function (Hawkins & Davis 2005, Iadecola 2004). A major component of this interface is the blood-brain barrier (BBB), which is formed by endothelial cells of the NVU. The specialized blood vessels of the BBB are characterized by the formation of tight junctions, expression of specialized transporters and pumps, and reduced basal endocytic activity as compared with vessels found in peripheral organs (Rubin & Staddon 1999). Although the barrier is generally termed the blood-brain barrier, there are in fact several distinct barriers between the periphery and the nervous system, including the blood-cerebral spinal uid barrier, the bloodretinal barrier, the bloodspinal cord barrier, and other barriers. Furthermore, this barrier is, in fact, a dynamic site of molecular transport that constantly regulates the ow of essential components, such as amino acids, across endothelial walls. Although many fundamental questions regarding BBB development and maintenance remain unanswered, the crucial
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role of the BBB in the protection and proper functioning of the nervous system has begun to attract the attention of researchers. Recent discoveries have uncovered several molecular cues that regulate barrier formation. For instance, the wingless-type protein (Wnt) signaling pathway was identied as a key activator of angiogenesis in the nervous system and subsequent BBB formation (Daneman et al. 2009, Liebner et al. 2008, Stenman et al. 2008). Notably, Wnt signaling has been extensively studied in nervous system development and function (Salinas & Zou 2008). The identication of Wnt signaling as a regulator of angiogenesis and BBB formation in the CNS provides molecular evidence to support the original observation that neuronal tissue provides instructive cues to advancing endothelial cells, forming specialized blood vessels in the nervous system (Stewart & Wiley 1981). These ndings are also another example of the common signaling pathways that drive the development of both the vascular and the nervous systems. Signicant progress has been made in identifying how vascular and nervous systems develop and function; recent advances highlight the common cellular and molecular mechanisms shared by these two systems. We examine these similarities and highlight the close relationship between the vascular and nervous systems, with an emphasis on their shared common developmental principles and how the interface between these systems, the BBB, is formed.
ANATOMICAL PATTERN OF THE VASCULAR AND NERVOUS SYSTEMS Evolution of the Nervous and Vascular Systems
The fundamental requirement for an organism to both sense its environment and broadly distribute nutrients throughout its body has driven the evolution of the nervous and vascular systems, respectively. Although the exact ori-
gin of the nervous system is debatable, investigators generally believe that modern nervous systems arose rst in the form of diffuse nerve nets, in sea-dwelling organisms such as cnidarians (Miller 2009). These organisms lack a vascular system (dened as a system to carry both oxygen and nutrients) because resources were readily available through diffusion and ingestion. On the basis of these observations, the general consensus indicates that the modern nervous system evolved prior to the vascular system. The major driving force behind vascular evolution is the need for oxygen to fuel aerobic metabolism in multicellular organisms, combined with the limited ability of oxygen to diffuse through tissue (Fisher & Burggren 2007). For arthropods, two systems seem to have evolved independently to distribute nutrients and oxygen. To deliver nutrients, hemolymph bathes all cells and is distributed by the coordinated movement of muscles and a primitive heart. Independent of nutrients, oxygen is delivered by the tracheal system. The tracheal system of insects is most akin to the modern vascular system as it relates to developmental principles, highlighted by the stereotyped formation of a tubular network that functions by directing air exchange of oxygen and carbon dioxide. In fact, many features of tube formation and sprouting are shared between the insect tracheal system and the modern vascular systems (Lubarsky & Krasnow 2003). As larger organisms evolved, however, a system to distribute oxygen and nutrients throughout dense tissue more efciently became necessary. Consistent with the chronology of neuronal and vascular evolution, several vascular-specic genes may have arisen later in evolution. Vascular endothelial growth factor (VEGF) was rst discovered as a growth factor that selectively mediates endothelial proliferation and vessel permeability (Keck et al. 1989, Leung et al. 1989), and expression of this hypoxiaresponsive gene is tightly regulated to control vasculogenesis and angiogenesis (Coultas et al. 2005, Fong 2009). Distant homologs to both mammalian VEGF and VEGFRs do exist
VEGF: vascular endothelial growth factor
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in invertebrates (Cho et al. 2002). The functions of these molecules in Drosophila are limited to controlling blood cell migration, a function that is speculated to be a precursor to the many VEGF-driven functions in mammals. One of the coreceptors for VEGF, neuropilin-1 (Nrp1), arose after the nervous system in evolution. Nrp1 was rst identied as a receptor for the axon guidance cue semaphorin (He & Tessier-Lavigne 1997, Kolodkin et al. 1997) and then subsequently shown also to be a receptor for VEGF (Soker et al. 1998). Nrp1 is found in vertebrates but is absent from invertebrates, whereas semaphorins and their other coreceptors, the plexins, are present in both invertebrates and vertebrates (Kolodkin 1996, Kolodkin et al. 1992, Tamagnone et al. 1999). Therefore, the evolution of the Nrp1 gene may have been congruent with the origin of the modern vascular system, yet Nrp1 is an essential component in vertebrate neuronal development as well. Nrp1 interaction with both semaphorin and VEGF is one example of how the vascular and nervous systems have co-opted the same molecular cascades for development. However, many of the guidance cues may have originated rst in the nervous system, with family members likely evolving later to play a role in vascular development. These pathways are discussed in more detail in subsequent sections.
Shared Anatomical Patterning

Similar patterning between the vascular and nervous systems is apparent at the anatomical level (Figure 1a). These similarities were likely rst observed at the macroscopic level (Vesalius
1543). With modern histological methodology, the coursing of artery and veins with nerve bundles can now be described at the microscopic level (Larriv ee et al. 2009, Mukouyama et al. 2002). How can such highly stereotypic patterning in two diverse systems be achieved? Do developing vessels provide signals for growing axons, or vice versa? Looking at the developing mouse embryo, one can see that vascular outgrowth precedes axon outgrowth (Figure 1b). Consistent with this observation, many molecular cues are provided by the developing vasculature to guide growing axons. For example, endothelin-3 is secreted by smooth-muscle cells of the external carotid artery, providing an attractive signal for extending axons of the superior cervical ganglia (Makita et al. 2008). Furthermore, vascular smooth-muscle expression of Artemin, a glial cellderived neurotrophic factor (GDNF) family member, is a signal attracting axons of sympathetic neurons (Honma et al. 2002). Artemin-decient mice show axon outgrowth and patterning defects, an observation that is phenocopied in mice lacking the Artemin receptor GFRalpha3. These ndings provide convincing evidence that vessels can direct neurons to form congruent patterns. As part of normal vascular and neural development, exuberant primitive networks are initially formed followed by the selective remodeling of these networks to form functional circuits. In the case of vascular remodeling, a robust vascular plexus is generated in the embryo, followed by remodeling to arteries, veins, and ne capillary networks. Vascular remodeling takes place after peripheral nerve development and is directed by peripheral sensory neurons and Schwann cells (Mukouyama et al. 2002).

Figure 1 Anatomical resemblance between the vascular and nervous systems. (a) Depiction of the shared anatomical patterning between nervous and vascular networks in the human body. Recent evidence indicates that blood vessels and neurons direct their outgrowth using shared molecular mechanisms, ultimately resulting in a similarly patterned network. (b) Although developing mouse embryos exhibit shared nervous and vascular patterning as well, the vascular system develops at a faster pace than does the nervous system. In fact, vessels can direct neurons to form congruent patterns. Left panel: neurons visualized by neurolament (Nf) staining of an E12.5 embryo. Right panel: blood vessels visualized by FLK1 in situ hybridization staining of an E10.5 embryo.
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Nf Nervous system Circulatory system
FLK1
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These conclusions are derived from both necessity and sufciency experiments. Mutant embryos that lack peripheral neurons do not undergo proper vascular remodeling, highlighted by defects in arterial differentiation. Moreover, mutants that misdirect peripheral axon outgrowth result in coalignment of blood vessels with these misguided neuronal patterns. Mukouyama et al. (2005) later showed that VEGF is one such cue involved in vascular remodeling and arteriogenesis that is provided by neurons and Schwann cells. More than four and a half centuries since Vesalius rst observed the overall anatomical similarities between vascular and neuronal networks, evidence now demonstrates that vessels and neurons codirect their patterning to achieve exquisite alignment at the anatomical level. For such guidance to take place, growing vessels and neurons must be able to sense the complex chemical cues that orchestrate the morphological changes necessary for proper patterning. These sensory mechanisms are found in the cellular structures that constitute the developing neuron and blood vessel.
SHARED CELLULAR MECHANISMS The Cellular Organization of the Nervous and Vascular Systems
In 1906, Camillo Golgi and Santiago Ramon y Cajal presented differing theories about the cellular organization of the nervous system in a famous Nobel debate. Golgi postulated that the nervous system consisted of a syncytial system with multiple cells of the nervous system possibly connecting via protoplasmic fusions to form a reticular system (Golgi 1906). Cajal, on the other hand, was an ardent proponent of the neuron doctrine, which argued that cells in the nervous system constitute individual components connected by a granular cement, or special conducting substance keeping neurons intimately in contact, yet in contiguity but not in continuity (Ramon y Cajal 1906). History has largely proven both Cajal
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and Waldeyer, the rst to propose the neuron doctrine, to be correct. We understand now that the nervous system consists of a complex network of diverse neurons, with extending axons connecting to dendrites of target cells via specialized contacts known as synapses. Many neurons must send axonal processes long distances through a labyrinth of tissue to reach their nal targets. The emergence of embryonic neurons from neuroblasts, followed by their migration and elaboration, results in single cellular units that possess the ability to receive and send information. The nervous system also contains a battery of glial cells, which are required for a multitude of nervous system functions, including enhancement of nerve conductance, immune surveillance, and neurotransmitter processing and signaling (Barres 2008). Golgi himself rst observed that glial cells make direct contacts with the vasculature, which were likely to be the astrocytic endfeet and their ensheathment of blood vessels (see The Neurovascular Unit, below). Oligodendrocytes and Schwann cells are responsible primarily for myelination of the central and peripheral nervous systems, respectively. They are functionally similar to pericytes and smooth-muscle cells of the vasculature because both are supporting cell types that enhance the function of the main constituent cell types, neurons and endothelial cells, respectively (Bergers & Song 2005, Betsholtz et al. 2005). However, it remains to be determined if similar signaling mechanisms are employed toward the formation of both endothelialpericytes and neuron-glia interactions. In contrast to network formation in the nervous system, which is the result of elaborated single neurons extending protoplasmic processes to form specic connections, the cellular components of the vascular system undergo blood vessel formation through two distinct processes. First, in vasculogenesis, endothelial cells differentiate from angioblasts to form a vascular plexus, which consists of a meshwork of connected strands of endothelial cells (Coultas et al. 2005). The vascular plexus serves as the starting substrate for further growth and
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renement. Second, new vessels sprout from existing vessels, a process termed angiogenesis. VEGF is necessary for both vasculogenesis and angiogenesis; however, other molecules provide additional cues for further differentiation and renement of the vascular system. Similar to the nervous system, where exuberant connectivity is followed by pruning to form a functional network, the vascular system also undergoes remodeling. This process is inuenced by both blood ow and contact with surrounding tissues. Furthermore, vessels formed via angiogenesis must sense their environment to follow the correct trajectory toward their nal target.
The Axon Growth Cone and Endothelial Tip Cell

Cajals illustrious career was also highlighted by his discovery of the axon growth cone, a
structure found at the tip of growing axons that guides outgrowth by sensing its local environment (de Castro et al. 2007). In Cajals own words, a growth cone is like a living batteringram, soft and exible, which advances mechanically, pushing aside the obstacles that it nds in its way until it reaches its peripheral destination (Ramon y Cajal 1917). Modern imaging has since veried Cajals assessment and has further dened the molecular components of the growth cone (Lowery & Van Vactor 2009). Specically, an axonal growth cone is found at the end of an axon shaft and consists largely of cytoskeletal machinery (Figure 2a). The hand-like structure itself is composed of both actin-rich lopodia and lamellopodia, with actin bundles that are connected to the microtubule framework that continues down the axon shaft. More than 100 years after Cajals original description of the axon growth cone, Gerhardt
Axon growth cone
Endothelial tip cell

Attractive guidance cues
a
Filopodium
Attractive guidance cues
Filopodium Lamellipodium Endothelial stalk cells
LamellIpodium
on Ax
Repulsive guidance cues
Figure 2
Capillary lumen (blood)
Endothelial tip cell Repulsive guidance cues
The axon growth cone and endothelial tip cell. Blood vessels and neurons must sense their environment to follow the correct trajectory toward their nal target. Cellular projections of axons and endothelia that guide such outgrowth share morphological features. (a) The axon growth cone forms at the leading edge of migrating axons through an elaborate cytoskeletal actin network linked to axonal microtubules. (b) The endothelial tip cell caps the migrating end of stalk cells, a lumenized column of cells that form a budding or angiogenic blood vessel. Although the tip cell is its own cellular entity separate from the rest of the growing blood vessel, it is the functional analog of the axonal growth cone. Both lopodial and lamellopodial projections of the extending growth cone and tip cell are believed to form the motor that physically pulls cells toward attractive cues ( green) and away from repulsive cues (red) as extending neurons and blood vessels sense their environment.
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et al. (2003) described a similar structure in angiogenic sprouts of growing blood vessels in the mouse retina. This structure is composed of a specialized cell found at the growing end of extending vessels, termed the endothelial tip cell (Figure 2b). The shaft of the extending vessel is composed of a lumenized endothelial cell chain made of stalk cells. From an architectural standpoint, the tip cell and associated endothelial stalk cells are similar to the axon growth cone and its associated axonal shaft. Tip cells, akin to axon growth cones, are actin-rich structures that dynamically navigate their environment when sprouting from existing vasculature. This process was elegantly observed through live imaging of green uorescent protein (GFP)-expressing vessels in transgenic zebrash, where extending tip cells were described as functionally similar to axon growth cones (Lawson & Weinstein 2002). Tip cells, at least in the retina, are exquisitely responsive to VEGF (Gerhardt et al. 2003). Using mice engineered to express distinct isoforms of VEGF that have different diffusion properties, investigators showed that endothelial tip cells migrate in response to VEGF gradients, whereas stalk cell proliferation is a property of total VEGF concentrations. These data are consistent with previous observations utilizing VEGF isoform mutants (Ruhrberg et al. 2002). Specically, a shallow VEGF gradient with high total concentrations of VEGF exposure results in large-diameter vessels with reduced branching. VEGF receptor 2 (Flk1) is both expressed on the tip cell and necessary for lopodia formation (Gerhardt et al. 2003). Thus, in the retina, VEGF acts as the predominant mitogenic factor and is chemoattractive for blood vessels. How is a tip cell selected to form an angiogenic sprout? The vascular system likely addressed this need by co-opting the Notch signaling cascade described rst in Drosophila. In the case of y tracheal development, a leading tracheal cell is selected by Notch-mediated lateral inhibition (Ghabrial & Krasnow 2006). In mammals, delta-like 4 (Dll4) appears to be the major Notch ligand regulating vascular
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development (Duarte et al. 2004, Gale et al. 2004, Krebs et al. 2004). Additional mechanistic studies have carefully dissected Dll4s role in vascular development and concluded that Dll4/Notch signaling is likely acting via lateral inhibition to establish the tip cell morphology, whereas adjacent cells are destined to become stalk cells (Hellstrom et al. 2007, Lobov et al. 2007, Suchting et al. 2007). These observations should not be surprising because Notch signaling is a major mechanism for cell fate determination in many tissues, including the nervous system (Gaiano & Fishell 2002). The cellular parallels between an extending neurons axon growth cone and an angiogenic blood vessels tip cell suggest that these structures may respond in ways similar to chemical cues provided by their respective environments. In 1963, Roger Sperry summarized the chemospecicity hypothesis, which postulates that chemical cues in the target tissue provide specic directions for the developing and regenerating nervous system, resulting in stereotyped neural network formation (Sperry 1963). Do blood vessels also respond to chemical cues resulting in stereotyped development? Recent discoveries have addressed this hypothesis at the molecular level and provide strong evidence that the nervous and vascular systems share common principles of development.
SHARED MOLECULAR MECHANISMS Axon Guidance Molecules and Angiogenesis

The possibility that the vascular and nervous systems respond to common molecular cues to guide their development is highly plausible owing to both their stereotyped circuitry and the shared common cellular structures used to sense their environment during development. We have described evidence that nerves may provide cues to growing vessels; likewise, the opposing relationship has also been established. A major theme of cellular guidance is the balance between attractive and repulsive forces
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(Figure 2). In introducing the endothelial tip cell, we have discussed how VEGF is a major attractive molecule for extending blood vessels (Gerhardt et al. 2003). Do other attractive cues exist for blood vessels? Are there repulsive forces that also guide developing vessels? In searching for additional candidate molecules, we now turn our attention to the accumulating evidence indicating that the classical axon guidance cues also guide vascular tip cells. Axon guidance molecules exist in four major families: (a) Slit/Robo, (b) semaphorin/ plexin/neuropilin, (c) Netrin/Unc5/DCC, and
(d) Ephrin/Eph (Figure 3). The role of these pathways in guidance of axons in the developing nervous system has been described extensively (Dickson 2002, ODonnell et al. 2009, Tessier-Lavigne & Goodman 1996). Recent studies also indicate that various well-described morphogens, including wingless-type proteins (Wnts), bone morphogenic proteins (BMPs), and sonic hedgehog (Shh) also mediate axon guidance (Butler & Dodd 2003, Charron et al. 2003, Lyuksyutova et al. 2003, Yoshikawa et al. 2003). With the exception of the Wnts, which are discussed in detail below related to
Vascular guidance molecules

EphrinB2 Other Ephrins Cell membrane Slit1-3 Sema3A, 3C, 3F
Class 4 and 5 Semas
VEGFs
Netrin1-4
Robo4
Robo1-3
Neuropilins
DCC
EphB4 PlexinD1
Other Plexins
VEGFRs
Unc5b
Other Ephs
Figure 3 Axon guidance pathways involved in angiogenesis. Schematic representation of the four major families of axon guidance ligand-receptor pairs: Slit/Robo, semaphorin/plexin/neuropilin, Netrin/Unc5/DCC, and Ephrin/Eph. Axonal guidance cues mediate complex cellular navigational programs within axons, as both chemoattractants and repellents. Recent evidence has identied roles for several of these classic guidance molecules in directing angiogenic tip cells toward their nal destination.
angiogenesis and BBB formation in the developing CNS, we discuss the role of the four major families of axon guidance molecules in angiogenesis.
The Slits and Robos in Vascular Guidance

The Slit/Robo family of axon guidance molecules was discovered as mediators of commissural axon crossing in Drosophila (Kidd et al. 1998, 1999; Seeger et al. 1993) and of axon guidance in C. elegans (Zallen et al. 1998). Wang et al. (1999) showed that Slit enhances axonal branching and elongation (Wang et al. 1999). There are three members of the Slit ligand family and four members of the Robo receptor family (Figure 3). Slits contain 4 leucine-rich repeats, 79 EGF repeats, and one laminin domain. The roundabout (Robo) receptors were named after their y loss-of-function phenotype of aberrant midline crossing, including axons repeatedly crossing the midline, giving the appearance of a circular road junction, or roundabout, when analyzed by histology (Seeger et al. 1993). The Robo1-3 receptors consist of ve Ig domains, three bronectin type 3 domains, and one intracellular signaling domain. A fourth member of the Robo receptor family, Robo4 or magic roundabout, contains only two Ig domains and two bronectin type 3 domains. Notably, Robo4 is almost exclusively expressed in the vasculature (Huminiecki et al. 2002). During neural development, Slit/Robo interactions can result in either a repulsive or an attractive response (Dickson & Gilestro 2006). Most data indicate that Slit/Robo binding on neurites primarily regulates a repulsive signal. Thus it was intriguing when Wang et al. (2003) found Slit2 to be an attractive factor for extending angiogenic sprouts through binding to Robo1 on endothelial cells. In contrast to this data, others have argued that Slit2 is repulsive to endothelial cells via binding to Robo4 (Park et al. 2003). To complicate matters further, it is unclear whether Slit2 binds to Robo4 because in vitro binding studies showed a convincing
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interaction only between Slit2 and Robo1, but not to Robo4 (Suchting et al. 2005). Robo4 may also mediate a repulsive or attractive response on the basis of cellular context, including cell-type and/or ligand binding. For example, Sheldon et al. (2009) recently showed that Robo1 and Robo4 can form heterodimers, indicating that Slit2 binding to Robo1 may result in signaling via Robo4. In addition to in vitro observations for Slit/Robo interactions in the context of the vascular system, studies in zebrash have shown that a knockdown of Robo4 results in an intersomitic vessel guidance defect with a reduction in vessel sprouting and a misdirection of those sprouts that do form (Bedell et al. 2005). However, no example of a vascular developmental defect in mouse knockouts of the various Slit/Robo genes has been reported. A recent study has proposed that Robo4 mediates vascular integrity in adult animals by inhibiting angiogenesis and reducing vascular permeability (Jones et al. 2008). Robo4-decient mice exhibited an increase in vascular sprouting and permeability in an oxygen-induced retinopathy model. Furthermore, injecting Slit2 reversed the increase in angiogenesis and vascular permeability in these models in a Robo4dependent fashion. These data support a repulsive role for Robo4 in angiogenic sprouting. However, because Robo4 is expressed on endothelial stalk cells in the retina, it is intriguing to speculate that Robo4 may inhibit new tip cell formation from existing vessels. To date, the interaction between Robo4 and Slit2 remains to be fully veried, leaving open the possibility that other ligands mediate Robo4 function.
The Semaphorins/Plexins/Nrps in Vascular Guidance

Although semaphorins (Sema) were rst discovered as mediators of axon growth cone collapse (Raper 2000), they are now more commonly recognized as proteins with robust effects on cellular morphology. There are eight classes of Sema proteins, including both membrane-bound and secreted forms. For the
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purposes of this review, we focus on one of the most versatile families, the class 3 semaphorins (Sema3). The Sema3 family members are secreted proteins that contain a Sema domain, a plexin/semaphorin/integrin domain (PSI), and an Ig domain (Figure 3). The Plexin and Neuropilin receptor families collaborate to form multimeric receptor assemblies responsible for downstream Sema signaling. The extracellular region of plexins consists of one Sema domain, three PSI domains, four Ig-like/plexins/transcription factor domains (IPT), and two Ig domains. A transmembrane helix links this region to the cytoplasmic region containing one coiled-coil helix and one Racbinding domain (RBD) that bisects a GTPase activating protein domain (GAP). Mammals have two neuropilin (Nrp) proteins: Nrp1 and Nrp2. Nrps share two complement-binding domains (CUB or a1a2 domains), two coagulation factor V/VIII homology domains (b1b2), and a meprin/A5 protein/phosphatase- domain (MAM) in the extracellular space followed by a transmembrane helix and a short cytoplasmic tail. Furthermore, Nrps can also be expressed as soluble isoforms lacking the MAM and transmembrane domains (Rossignol et al. 2000). In addition to being semaphorin receptors, Nrps bind multiple VEGF isoforms and mediate distinct VEGF functions (Pan et al. 2007; Soker et al. 1998, 2002). The Sema/Plexin/Nrp family is a prime example of a group of molecules with diverse functions in both neuronal and vascular development. Here, we focus on their roles in vascular biology. Sema3s were originally classied as potent axonal chemorepellents that signal indirectly through Plexin receptors by binding to Nrps (Chen et al. 1997, Fujisawa 2004, He & TessierLavigne 1997, Kolodkin et al. 1997). Of the seven Sema3s (A-G) reported in mammals, several function in more versatile ways, including a key role in guiding extending endothelial cells (Tran et al. 2007). Sema3A was rst described in the vascular system as a mediator of vessel tip cell lamellipodial collapse and was thus an
inhibitor of angiogenesis (Miao et al. 1999, Serini et al. 2003, Shoji et al. 2003). However, a role for Sema3A in endothelial biology and vascular development has been disputed. More recent studies have shown that Sema3A does not reduce endothelial cell migration (Pan et al. 2007), nor does it compete with VEGF for binding to Nrp1 (Appleton et al. 2007). Furthermore, sema3a/ knockout mice do not have vascular development phenotypes (Vieira et al. 2007). Additional studies may be needed to determine if Sema3A plays a more specic or subtle role in vascular biology that has yet to be discovered. For example, although the overwhelming evidence suggests that semaphorins act as chemorepulsive cues in the nervous system, at least one example shows that the cellular context can switch Sema from a repulsive to an attractive signal (Song et al. 1998). In contrast to Sema3A, Sema3E is emerging as a critical mediator of vascular biology through its direct interaction with the endothelial expressed receptor, PlexinD1 (Gitler et al. 2004, Gu et al. 2005, Torres-Vazquez et al. 2004). The vascular expression of plexinD1 was rst described in two separate studies, one examining all Plexins and focusing on their roles in the nervous system (Cheng et al. 2001) and the other looking specically at plexinD1 expression during embryonic development (van der Zwaag et al. 2002). Sema3e and plexinD1 loss-of-function studies in both zebrash and mouse result in a vessel guidance defect consistent with a repulsive role for Sema3E acting via PlexinD1. Somites express Sema3E, thereby preventing the PlexinD1expressing intersomitic vessels from entering the somites. However, plexinD1 knockout mice show a cardiac development defect not observed in sema3E knockouts, thus suggesting that another Sema, speculated to be Sema3C, may act through PlexinD1. Nrp receptors bind both VEGF and class 3 semaphorins. It is therefore not surprising that knockouts of both Nrp1 and Nrp2, which are expressed in developing vasculature and lymphatic systems respectively, lead to numerous vessel-patterning and lymphangiogenic
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defects (Gu et al. 2003, Kawasaki et al. 1999, Takashima et al. 2002). However, Nrps interactions with both VEGFs and Sema3s complicate the interpretation of these results, making it difcult to pinpoint a particular phenotype as being related to Sema or VEGF. To address this issue Gu and colleagues (2002) rst performed structure/function studies to identify residues responsible for Sema3 binding to Nrp1. Subsequently, knock-in mice expressing mutant Nrp1 lacking Sema binding and keeping VEGF binding intact were generated (Gu et al. 2003). These mice showed no vascular defects, suggesting that Nrp1s interaction with VEGF, but not Sema, is critical for vascular patterning. On the basis of Nrp1s multifaceted role in vascular biology, this molecule is now being therapeutically targeted to modulate angiogenesis in vivo.
The Netrin/Unc5/DCC Family in Vascular Guidance

Netrin was rst identied as UNC-6 in C. elegans (Ishii et al. 1992), and three Netrin family members have been identied in mammals (Moore et al. 2007). The netrins are secreted proteins that contain one laminin domain, three EGF-like repeats, and a carboxylterminal domain (CRD) (Figure 3). Netrins act as both chemoattractants and chemorepellents using their interactions with respective receptors. During CNS development, Netrin1 is secreted from cells at the ventral midline to attract commissural axons (Kennedy et al. 1994, Serani et al. 1994) via activation of the DCC (Deleted in Colorectal Cancer) receptor (Keino-Masu et al. 1996). In netrin1 and dcc knockout mice, most commissural axons fail to reach and cross the midline (Fazeli et al. 1997, Serani et al. 1996). DCC and neogenin are members of the DCC family of receptors and contain four Ig domains, six Fn3 domains, and one transmembrane domain, followed by an intracellular signaling domain with putative protein interaction and phosphorylation sites (Moore et al. 2007). DCC can also interact with Robo
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receptors to inactivate Netrins attractant activity once midline crossing has been achieved (Stein & Tessier-Lavigne 2001). Conversely, Netrin-mediated axon repulsion involves the activation of Unc5 (uncoordinated 5) receptor family members, either as homodimers or Unc5-DCC heterodimers (Hong et al. 1999, Keleman & Dickson 2001, Leonardo et al. 1997). The Unc5 family contains two Ig domains, two thrombospondin type I domains, one transmembrane domain, one Zona occludens-5 domain, one DCC interacting domain, and one death domain. Netrins and their cognate receptors have also been implicated in the regulation of several developmental processes in nonneuronal tissues. The rst hints that Netrin/Unc5 signaling may regulate blood vessel morphogenesis were discovered as a consequence of knocking out the Unc5b receptor (Lu et al. 2004). Depletion of Unc5b in mice and zebrash resulted in excessive tip cell lopodia formation and branching, suggesting that Unc5b activation drives endothelial repulsion. Investigators showed that Unc5b is present in the tip cell of extending blood vessels (Bouvr ee et al. 2008, Larriv ee et al. 2007). Knockdown of netrin1a in zebrash results in a loss-of-function phenotype similar to the unc5b loss-of-function. Similar to Sema3A and PlexinD1, intersomitic paths express Netrin1a and thereby prevent the Unc5b-expressing tip cells from meandering off path into the somites. In mice, the ligand for Unc5b-mediated vessel tip cell repulsion remains to be identied because netrin1 knockouts maintain normal vascular architecture (Salminen et al. 2000, Serani et al. 1996). The lack of Netrin1 involvement in mammalian blood vessel repulsion may be resolved by ndings involving the Netrin4 family member. Netrin4 was upregulated in angiogenic endothelium and bound Unc5 receptors, possibly through direct interactions with the coreceptor Neogenin (Lejmi et al. 2008, Qin et al. 2007). Although in vivo mammalian loss-of-function data are lacking, recent in vitro endothelial migration results suggest that both Netrin1
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and Netrin4 can inhibit VEGF-induced angiogenesis. Conversely, Netrin1- and Netrin4-mediated blood vessel chemoattraction is suggested on the basis of both in vitro vessel proliferation and migration studies and in vivo viral delivery studies. Nevertheless, the cognate in vivo receptors for Netrin1 and Netrin4 remain to be conclusively identied. In addition, these data remain controversial because knockout phenotypes for Unc5b reported by various other groups do not mimic those seen in these studies (Nguyen & Cai 2006, Park et al. 2004, Wilson et al. 2006). Moreover, the functional duality shared by many axon and vessel guidance cues suggests that Netrin/Unc5/DCC may mediate both repulsion and attraction in the vascular system (Yang et al. 2007). Thus, discovering the vascular in vivo receptor(s) for Netrin1 and Netrin4 and the ligand for Unc5b may help to clarify the existing data.
The Ephrin/Eph Family in Vascular Guidance

The Ephrin/Eph receptor tyrosine kinase family of short-range axon guidance molecules was rst identied as chemorepellents (Cheng et al. 1995, Drescher et al. 1995). Their dual functional nature was subsequently elaborated, whereby Ephrin/Eph interactions serve as both attractive and repulsive cues in both the nervous and the vascular systems (Palmer & Klein 2003). Ephrin ligands contain one cupredoxin homolog domain (Figure 3), whereas EphrinA1-5 are anchored to plasma membranes via a glycosyl-phosphatidyl-inositol moiety and EphrinB1-3 have a transmembrane domain followed by a cytoplasmic tail containing a PDZ binding domain that can mediate reverse signaling (Holland et al. 1996). The Eph receptors include 13 family members: EphA1-8, EphB1-4, and EphB6. Eph receptor tyrosine kinases contain one laminin domain, two tumor necrosis factor receptor (TNFR) CRD domains, two Fn3 domains, a transmembrane domain followed by a kinase domain, and a sterile alpha motif (SAM)
domain. In the nervous system, these molecules drive many wiring processes, including midline pathnding, dendritic spine formation, and synaptic plasticity (Palmer & Klein 2003). Ephrin/Eph signaling molecules also control blood vessel development. Ephrin/Eph participate in another key aspect of vascular development: the maintenance of vascular patterning through segregated expression in venous and arteriole vessels. EphB4 is expressed in developing veins, whereas EphrinB2 is found in arteries, providing a key mechanism for repulsive tissue boundary preservation within vascular networks (Adams et al. 1999, Gerety et al. 1999, Wang et al. 1998). These observations are best depicted in ephrinB2 knockout mice, in which angiogenesis defects in both embryonic arteries and veins suggest that both forward and reverse Ephrin/Eph signaling takes place in developing vasculature. Conversely, Ephrin/Eph guidance factors function as positive cues in the context of tumor angiogenesis. For example, EphrinA1 stimulates angiogenesis when expressed on tumor cells, and treatment with the EphA2 receptor ectodomain inhibits neovascularization in these tumor models (Ogawa et al. 2000, Pandey et al. 1995). Therefore, like the other axon guidance family members described, the Ephrin/Eph family of vascular guidance cues also exhibits exquisite dynamic versatility, mediating complex cellular navigational programs within axons and blood vessels as both chemoattractants and repellents. The past decade of discoveries has elucidated key roles for axon guidance molecules in vascular development and has shown that they utilize biological principles similar to those rst observed in the nervous system. These pathways are now a focus of research in the disease setting, for modulating both blood vessel and axonal growth (see sidebar, Axon Guidance Molecules as Targets for Cancer). Having focused on how the vascular and nervous systems share common cues for development, we now turn our attention to the interaction between the vascular and the nervous systems, again with an emphasis on development.
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AXON GUIDANCE MOLECULES AS TARGETS FOR CANCER

As solid tumors begin to grow and become hypoxic, tumor cells recruit blood vessels by secreting VEGF and other angiogenic factors. Blocking angiogenesis is now well established as a mechanism to treat cancer; this process slows tumor growth by reducing nutrient and oxygen transfer to tumor cells (Ferrara & Kerbel 2005). With the discovery of numerous axon guidance molecules playing an essential role in vessel growth during development, investigators have proposed that these mediators of angiogenesis represent a new set of potential cancer targets. Furthermore, several axon guidance molecules have also been studied in the context of tumor cell biology, for example in mediating tumor cell migration. Future studies in both animal models and cancer patients will determine the value of targeting axon guidance molecules as a means to slow tumor growth and disease progression.
vascular network within an even more intricate neural network. Before describing what is known about vascular development in the CNS we rst illustrate in detail the properties of the interface between the vascular and nervous systems, with a focus on the BBB, an essential component of the multicellular NVU.
Molecular Properties of the Blood-Brain Barrier

The now classic experiments demonstrating that a polar dye, trypan blue, injected into blood is specically excluded from the CNS serve as the rst known experimental paradigm in which a barrier between the CNS and peripheral tissue was described (Goldmann 1909, 1913). In the rst set of experiments, investigators prematurely concluded that brain tissue had minimal dye-binding afnity compared with other non-CNS organs. The presence of a BBB was subsequently established with the converse experiment of dye injection into the cerebrospinal uid, the result being staining of brain. Taken together, investigators concluded that various molecules, in this case typan blue (872.88 Da), are uniquely excluded from the CNS and thus that a biological barrier must be in place. The BBB is an essential regulator of bloodbrain exchange of nutrients and waste that operates on several distinct levels (Figure 4). First, endothelial intercellular assemblies such as tight and adherens junctions function as a physical barrier that restricts the paracellular movement of molecules across the BBB.
THE INTERFACE BETWEEN THE VASCULAR AND NERVOUS SYSTEMS

The human brain features a large vascular network of 400 miles of blood vessels that receives more than 20% of the bodys cardiac output and utilizes most of its blood glucose (Begley & Brightman 2003). These outstanding characteristics underscore the evolved importance of efcient and thorough exchange of both nutrients and waste between blood and the CNS. This architecture also represents a daunting task for development of a complex

Figure 4 The cellular and molecular properties of the blood-brain barrier (BBB). Many proteins function at the blood-brain barrier (BBB) to regulate molecular exchange. (a) These components include small molecule transporters (P-gp/MRPs, amino-acid, and glucose transporters), large molecule transporters (insulin, LDL, transferrin, and leptin receptors), junctional assemblies (tight and adherens junctions), and metabolic enzymes (P450-related, MAO, and GGT). Tight junctions at the BBB limit most paracellular movement and are composed of several protein families, including occludin, claudin, e-cadherin, ZO, JAM, catenins, cingulin, and actin. (b) Small and/or lipophilic molecules can bypass the tight junctions but are rapidly degraded by intracellular enzymes and actively pumped back into the bloodstream by P-gp and other multidrug-resistant proteins (MRPs). Large molecule, amino-acid, and glucose transporters allow regulated passage of essential nutrients from blood to brain. (c) Electron micrograph of endothelial contacts within the blood-brain barrier (image courtesy of Reese & Karnovsky 1967). Arrows mark intercellular kissing-points indicative of tight junctions.
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BBB: tight junctions, transporters, and transcytosis
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Second, several transport mechanisms resident to the BBB jettison unwanted molecules out of the CNS while importing molecules essential for proper brain function. Third, endothelial cells of the BBB express a variety of enzymes that can metabolize unwanted molecules, including toxins (de Boer et al. 2003, Iadecola 2004, Reese & Karnovsky 1967, Rubin & Staddon 1999, Zlokovic 2008). The molecular exchange across the endothelium of the BBB is limited and tightly regulated. Most large, polar metabolites are excluded from blood-brain movement through a complex network of tight junctions and adherens junctions (Figure 4). Integral membrane proteins, such as occludin and claudins, physically associate with the actin cytoskeletal network through zonula occluden adaptor proteins. Claudin-5 is the most well characterized TJ protein within the BBB. It is ubiquitously expressed in all tissue vasculature, and deletion results in leakage of small molecules across the BBB and early postnatal lethality (Nitta et al. 2003). Although cell culture experiments demonstrate that truncated occludin expression functionally compromises the physical barrier because of low transcellular electrical resistance and increased leakage of small molecules across the BBB (Bamforth et al. 1999), occludin is not required for TJ structural formation in vivo (Saitou et al. 2000). Although the BBB is a highly effective gatekeeper, substances that are either lipophilic or smaller than 400 Da may effectively bypass the physical barrier. The metabolic barrier eliminates such chemicals that would otherwise move from the blood into the CNS. As the primary route of drug metabolism, p450-related cytochrome enzyme oxidizes unwanted substances within the cytoplasm of endothelial cells (Alavijeh et al. 2005). Likewise, monoamine oxidase (MAO) also contributes to the metabolic barrier to protect the brain from circulating neurotoxins and biogenic amines. For instance, MAO metabolizes the neurotoxin MPTP and attenuates the parkinsonism associated with this drug (Kalaria & Harik 1987). In some cases, enzymatic activity within the BBB does not
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remove unwanted molecules but instead facilitates the transport of essential substances from blood to brain. Through its transpeptidase activity, gamma-glutamyl transpeptidase (GGT) assists in the transfer of amino acids across the BBB (Hawkins et al. 2006). By modulating pyroglutamate levels, GGT indirectly stimulates a wide array of sodium-dependent amino acid transporters at the plasma membrane (Figure 4). Amino acids (AA) represent one of many classes of nutrients essential for proper CNS function and cannot passively diffuse across the BBB because they are neither lipophilic nor smaller than 400 Da. Transport mechanisms tightly facilitate the regulated exchange of these substances across the BBB by utilizing a diverse array of membrane protein carriers and transcytotic receptors. Small metabolites including amino acids, glucose, nucleosides, and vitamins typically move down concentration gradients in the direction from blood to brain through carrier-mediated transporters. GLUT1, a member of the sodium-independent class of glucose carriers, shuttles glucose into the brain and is highly expressed in the BBB. The neurodevelopmental disorder known as GLUT1 deciency syndrome is caused by an autosomal dominant mutation of the glucose transporter and is manifest by seizures and reduced head growth after birth (Wang et al. 2005). These genetic ndings likely underscore the critical importance of proper exchange of nutrients, particularly glucose, across the BBB. The decits observed in humans with this disorder can be modeled in mice that have lost one copy of Glut1 (Wang et al. 2006). Conversely, a subclass of transporters called sodium-dependent AA transporters helps prevent the accumulation of potentially neurotoxic excitatory AAs such as glutamate and aspartate (Zlotnik et al. 2007). Even trace amounts of metabolites that can passively diffuse across the plasma membrane can be highly neuroactive. In these cases, active efux transport mechanisms are necessary to drive molecular exchange against concentration gradients from brain to blood (Figure 4b). The
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most well-characterized efux conduit is the multidrug resistance protein P-glycoprotein, one of several multidrug resistance-associated proteins (MRPs). This membrane protein efciently removes a diverse structural array of lipophilic molecules through an internal pore (Aller et al. 2009). Researchers propose that through the combined action of several MRPs, both potentially benecial therapeutics and neurotoxic chemicals are rapidly cleared from the brain (Hermann & Bassetti 2007, Loscher & Potschka 2005). Receptor-mediated transcytosis is responsible for the movement of large proteins across the BBB (Pardridge 1988). Leptin, insulin, transferrin, and low-density lipoproteins (LDL) are a few examples of proteins that gain entry into the CNS through binding to their cognate receptors within BBB endothelium and are internalized via endocytic vesicles (Figure 4b). This biological transport is termed receptor-mediated transcytosis. How directionality is achieved when transporting these large proteins across the BBB is not well understood. We presume that the polarity of BBB endothelium and differences between CNS and blood environments drive the transport of these molecules in a direction that benets the organism.
The Neurovascular Unit

For the past two decades, studies have thoroughly established that BBB development, regulation, and maintenance are controlled by various cells in the brain, including pericytes, astrocytes, and neurons (Ek et al. 2006, Janzer & Raff 1987, Stewart & Wiley 1981). The close juxtaposition and physical contact between these different cell types and endothelial cells form the neurovascular unit (NVU) (Figure 5). This functional unit enables efcient oxygen and nutrient ow between various cell types in the CNS, which promotes proper brain function (Banerjee & Bhat 2007, Iadecola 2004). Intercellular communication within the NVU allows the CNS to achieve tight temporal control of cerebral blood ow to
match metabolic needs of the surrounding neural tissue. During development, the neuroepithelium and pericytes actively regulate angiogenesis and vessel guidance. For example, neurons secrete proangiogenic factors such as VEGF (Haigh et al. 2003, Raab et al. 2004) and Wnt ligands (see below) to provide growth and differentiation signals to extending blood vessels. In the mature NVU, neurons further regulate vascular function via signaling through astrocytes, whereas smooth-muscle cells and pericytes control blood ow in response to this neuronal activity. We recognize that cerebral blood ow is regulated by neuronal activity, but the exact mechanisms of NVU function are still under investigation. The functioning of the NVU has been addressed in detail elsewhere (Banerjee & Bhat 2007, Iadecola 2004). Here we address the development of the NVU and its major component, the BBB. In an elegant study utilizing the morphological differences between chick and quail endothelial cells, the unique features of BBB vasculature were shown to be extrinsically induced by the CNS microenvironment (Stewart & Wiley 1981). These studies demonstrated that avascular embryonic quail brain transplanted into chick gut is vascularized by chick peripheral vessels. These vessels take on features of the BBB including tight junctions and the ability to restrict molecular diffusion into the CNS tissue, thus strongly indicating that instructive cues are produced by neuroepithelium to drive barrier formation. It has also become increasingly clear that the barrier is established during the earliest stages of angiogenic development, while astrocytes further drive BBB maturation (Ek et al. 2006, Janzer & Raff 1987). Pericytes and neurons of the NVU are present at the onset of CNS angiogenesis and BBB formation, consistent with the idea that many features of the BBB are initiated during vascular sprouting into the CNS (Virgintino et al. 2007). Furthermore, hallmark BBB components such as Claudin-5 and GLUT1 are present at the earliest stages of CNS angiogenesis; newly formed vessels are established with a physical barrier that can exclude small molecules from entering
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the embryonic CNS (Daneman et al. 2009, Ek et al. 2006). Whereas CNS angiogenesis and BBB formation occur during embryogenesis, formation
of astrocytes and their subsequent ensheathment of vessels start postnatally in rodents, thus the NVU does not fully form until after birth. Detailed rat studies have observed complete
The neurovascular unit (NVU)
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encirclement of vessels by astrocytes three weeks after birth (Caley & Maxwell 1970). Astrocytic regulation of BBB function has been characterized through in vitro coculture experiments and CNS disease studies (Argaw et al. 2009, Hurwitz et al. 1993, Janzer & Raff 1987). However, how astrocytic endfeet affect specic BBB properties during maturation remains to be determined. For example, astrocyte removal had no effect on the physical barrier of the BBB when using the tracer horseradish peroxidase (HRP) to investigate integrity (Krum & Rosenstein 1993). Substantial progress has been made on understanding how the NVU functions in the adult animal (Banerjee & Bhat 2007, Iadecola 2004). Growing evidence also indicates that the dysfunction of the NVU may be a major contributor to neurological disease (Iadecola 2004, Neuwelt et al. 2008, Zlokovic 2008). In particular, altered cerebral blood ow and a compromised BBB have been described in numerous neurodegenerative conditions (see sidebar, The BBB and Neurodegenerative Disease). Nevertheless, to gain further insight into the function of the NVU and associated BBB, a better understanding of the development of these systems must be established.
THE BBB AND NEURODEGENERATIVE DISEASE

In addition to the neuronal loss and inammatory processes that are common hallmarks of neurodegenerative disease, accumulating evidence indicates that the BBB is disrupted in many neurodegenerative diseases, including Alzheimers, Parkinsons, and ALS (Zlokovic 2008). It remains to be seen if the disruption of the BBB is a cause or consequence of neurodegenerative processes. Nevertheless, the fact that the BBB is disrupted is pushing researchers to understand BBB biology in the context of neurodegenerative disease, addressing the following questions, among others: Which molecular mechanisms drive barrier disruption? How does disruption of neurovascular coupling alter CNS function? Is drug delivery altered in neurodegenerative disease? And ultimately, will repairing the BBB slow neurodegenerative disease? This area of research is ripe for discovery and may lead to the development of therapeutics with a primary mechanism of barrier modulation to treat these diseases.
CNS Angiogenesis and BBB Formation: A Role for Wnt Signaling

The canonical Wnt signaling pathway was recently identied as an essential regulator of CNS angiogenesis and BBB development (Daneman et al. 2009, Liebner et al. 2008, Stenman et al. 2008). This developmental
pathway involves Wnt ligand-driven activation of Frizzled and LRP cell surface coreceptors, which then stabilize cellular betacatenin through activated Disheveled protein (Figure 6). The increase in beta-catenin levels then induces transcription of Wnt-responsive genes through Lef-1/TCF DNA binding proteins (van Amerongen & Nusse 2009). In situ hybridization experiments found that the Wnt ligands, wnt7a and wnt7b, are expressed in neural progenitor cells during CNS angiogenesis (Figure 6a). Functionally, Wnt7a/7b were required for developmental angiogenesis induced from the vascular plexus surrounding the spinal cord. Invading vessels utilize

Figure 5 The neurovascular unit (NVU). (a) The mature blood-brain barrier consists of close association and physical contact between endothelial cells, neurons, astrocytic endfeet, basal lamina, and pericytes. The NVU enables efcient cellular communication for proper brain function, such as regulating cerebral blood ow in response to neural activity. (b) Immunohistochemical staining of the cellular components within the mouse NVU. Antibody staining against BBB transporters P-gp and GLUT1 label endothelial cells, whereas NG2 detects the pericytes found along these endothelial cells, which tightly regulate blood ow. Note the tight ensheathment of endothelial cells by the basal lamina (Collagen-IV) and astrocytes (GFAP) visualized by costaining. Astrocyte endfeet (GFAP), in concert with connections from interneurons, regulate neurovascular calcium signaling, blood ow, ionic transport, and water transport.
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Figure 6 Wnts regulate angiogenesis and BBB formation in the nervous system. The canonical Wnt signaling pathway is essential for proper BBB development. (a) Wnt ligands 7a and 7b are specically expressed in neuroepithelium to induce developmental angiogenesis from the perineural vascular plexus into the developing neural tube. These secreted proteins may form attractive gradients that directly shape endothelial tip cell morphology. Furthermore, Wnt signaling in the CNS drives BBB endothelial cell differentiation, thereby inducing glucose transporter-1 (GLUT1) and tight junction protein Claudin-3 expression. (b) The specicity of canonical Wnt signaling on CNS vasculature is highlighted by the observation that Wnt-responsive genes are enriched in BBB endothelium when compared with nonbrain endothelium. This developmental pathway involves Wnt liganddriven activation of Frizzled and LRP cell surface coreceptors, which then activate Dishevelled. This protein inactivates GSK-3 activity in complex with APC and Axin, thereby stabilizing beta-catenin to drive Wnt target genes through Lef-1/TCF DNA binding proteins.
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paracrine signaling derived from the neuroepithelium to coordinate proper BBB development because loss-of-function of either wnt7a/7b in neuroepithelium or of beta-catenin in endothelial cells leads to angiogenic defects and subsequent vascular hemorrhage. These mice exhibit angiogenic defects only in the CNS and not in the periphery, consistent with the observation that Wnt signaling is not activated in non-CNS endothelium (Figure 6b). Although BBB formation parallels angiogenesis in the CNS, proper CNS vascular sprouting alone is not sufcient for complete barrier development. BBB formation requires the expression of a diverse array of gene products to establish barrier function. As previously discussed, GLUT1 is essential for proper brain development because autosomal dominant mutations lead to several neurological maladies and developmental delays (Wang et al. 2005). The tight junction protein Claudin-3, also found predominantly in the BBB vasculature, has been linked to BBB decit under pathological conditions as well (Wolburg et al. 2003). Wnt signaling appears to induce not only CNS angiogenesis, but also expression of BBB components GLUT1 and Claudin-3 (Daneman et al. 2009, Liebner et al. 2008, Stenman et al. 2008). Either Wnt7a/7b or beta-catenin depletion results in a lack of GLUT1 and Claudin-3 expression at the BBB. Conversely, ectopic Wnt7a/7b expression induced GLUT1 expression outside the CNS, whereas addition of Wnt ligands to cultured brain endothelial cells induced both GLUT1 and Claudin-3 protein expression. These ndings emphasize the dual functional nature of Wnt-mediated vascular development in the CNS, whereby both angiogenesis and BBB formation are tightly coupled. We do not yet know if Wnt also plays a role in the nal maturation of the NVU, including driving astrocytic associations with CNS blood vessels. Although the ligands required for Wntdriven BBB development have been at least partially identied as Wnt7a/7b, the cognate receptor(s) have not. Most members of the Frizzled family (Fzd3, 4, 6, 8, 10) and both Lrp5 and Lrp6 are expressed in the CNS.
Daneman and colleagues (2009) speculate that Fzd8 may be important in CNS angiogenesis and BBB development on the basis of its specic enrichment in CNS vasculature. The molecular mechanisms of Wnt-driven BBB development in the CNS could be further elucidated by examining whether endothelial-specic deletion of particular Fzd or LRP isoforms can mimic defects observed in Wnt7a/7b knockout embryos. The specicity of Wnt signaling on CNS vasculature is also highlighted by the observation that canonical Wnt signaling components, such as Lef-1, Tcf7, Axin2, Apcdd1, Ppard, Tbx3, Foxf2, and Stra6, are enriched in BBB endothelium when compared with peripheral tissue endothelium (Daneman et al. 2009). Because this Wnt expression prole was observed in adult mice, the tissue specicity of canonical Wnt signaling appears to be maintained in mature BBB. Could this result indicate that chronic Wnt/beta-catenin signaling is required to maintain proper BBB function within the NVU in the mature animal? The fact that neurons remain a consistent source of Wnt ligand from embryo to adulthood supports such a possibility. For instance, Wnt7a/7b are actively secreted during synaptic activity, which then mediates experience-related regulation of synapse abundance and network complexity (Gogolla et al. 2009). A role for Wnt signaling in BBB maintenance is particularly attractive because it provides a new drug target for both selective opening of the barrier for entry of CNS therapeutics and selective tightening of the BBB in CNS diseases where barrier function may be compromised (Zlokovic 2008). Liebner and colleagues (2008) suggest that Wnt signaling activity is low by the time the BBB is fully mature on the basis of the observation that Wnt signaling activity per unit vessel length shows a precipitous drop after embryonic day 15.5. However, this result could be reective of endothelial cells lengthening dramatically during development, such that the number of nuclei per unit length drops (Liebner et al. 2008). Therefore, BBB function will have to be examined upon suppression of
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Wnt/beta-catenin signaling in the adult animal to determine the validity of this model. In hindsight, the Wnt signaling pathways involvement in BBB development within the CNS should not have come as a surprise. In the developing mouse, Wnt gradients direct axonal pathnding, regulate synaptogenesis, and coordinate axon and dendrite development (Salinas & Zou 2008). Thus the theme of CNS guidance cues regulating vascular development is once again apparent. Several other morphogenic processes including the BMP and hedgehog signaling pathways act in concert with the Wnt pathway in the CNS (Charron & Tessier-Lavigne 2005). Specically, overlapping BMP and Shh ligand gradients cooperate with Netrin1 to drive commissural axons from the roofplate down to the oorplate of the developing spinal cord (Augsburger et al. 1999, Charron et al. 2003). Overlapping Shh and Wnt4 gradients subsequently direct these axonal projections along the anterior/posterior axis to the nal target tissue. Could these signaling pathways be required for proper CNS angiogenesis and BBB development as well? There is precedence for not only shared ligandreceptor paracrine signaling driving both neuronal and vascular patterning, but also Wnt morphogen involvement in CNS angiogenesis and BBB formation, as just described. Indeed, Araya et al. (2008) demonstrated that brain endothelial cells secrete BMP ligands, resulting in astrocyte-derived VEGF production and subsequent BBB breakdown. In addition to the morphogenic signaling pathways, a wide array of axon guidance molecules are also potential candidates for CNS-specic angiogenic and BBB formation cues. Notably, Nrp1 is required for angiogenic sprouting in the CNS (Gerhardt et al. 2004). We need to identify which, if any, of the many axon guidance cues that have been implicated in vessel guidance, branching, development, or maintenance also contribute to CNS angiogenesis and BBB development. The rhodopsin-family G proteincoupled receptor (GPCR), Moody, was recently shown to be required for formation and maintenance
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of the Drosophila hemolymph-brain barrier, the functional analog of the mammalian BBB (Bainton et al. 2005). Does GPCR signaling control BBB permeability in mammalian systems as well? Although many GPCRs are expressed in the mouse brain, there are no obvious mammalian orthologs to Moody that can be identied using sequence homology. Nevertheless, it is interesting to note that the adhesion GPCR, gpr125, is upregulated following brain injury and is specically expressed in lung and cortical tissue (Pickering et al. 2008). In addition, the vasodilator bradykinin acutely modulates BBB permeability through its cognate GPCRs, bdkrb1 and b2 (Bartus et al. 1996, Su et al. 2009). We are only just beginning to understand the signaling pathways that coordinate the formation of a fully functional BBB. Furthermore, specic pathways may drive BBB development within different regions of the CNS. Consistent with this possibility, some evidence indicates that Wnt/beta-catenin signaling is not necessary for pan-CNS angiogenesis and BBB formation. For instance, Wnt7a/7b ligands are not expressed in dorsal neural tubes, and as a consequence, Wnt7a/7b knockout mice can still form proper blood vessels in this region (Stenman et al. 2008). Similarly, beta-catenin knockouts exhibit normal BBB formation in the hindbrain (Daneman et al. 2009), and Fzd4 knockout mice show barrier decits in the cerebellum, but not the cortex, suggesting that multiple Fzd receptors may coordinate BBB development in a region-specic fashion (Ye et al. 2009). Therefore, unique positions within the CNS seem to require activation of other signaling pathways. Unbiased approaches toward uncovering novel molecular mechanisms of BBB development and maintenance are needed at this juncture. Signaling pathways coregulated with Wnt activation in CNS endothelium may suggest participation in BBB formation. Downstream of Wnt signaling, many of the BBB-related genes directly or indirectly regulated by Wnt/beta-catenin have yet to be identied. Investigation of BBB endothelial cell expression
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proles along the developmental timeline may paint a more comprehensive picture of BBB development, maturation, and maintenance.
CONCLUSIONS AND FUTURE DIRECTIONS

Mirroring the sequential origin of the nervous and vascular systems, researchers have applied principles of nervous system development to address vascular biology questions. This approach stemmed from the original observation that the nervous and vascular systems share anatomical and cellular similarities. Moreover, both systems must be present throughout an organism to deliver and receive vital information and nutrients. It is apparent now that similar molecular mechanisms drive both vascular and neuronal network formation. These discoveries have been key to accelerating our understanding of how blood vessels develop. Also, the vascular system can directly impact nervous system development and vice versa; thus caution should be taken when characterizing phenotypes after disrupting signaling pathways in either system. We have described examples of specic axon guidance molecules regulating vascular biology. Although not comprehensive, these examples illustrate the inuence that discoveries in the eld of axon guidance have had on understanding vascular development. However, much remains unknown. For instance, the role that VEGF plays in neural development and function is just beginning to be unraveled (Ruiz de Almodovar et al. 2009). In addition, we have not addressed in this review the close relationship between blood vessels and neural progenitor cells at the vascular niche (Barami 2008). Many important questions remain that would further our understanding of the relationship between vascular and nervous system development. Are there other guidance cues that mediate both vascular and neural development? How are developmentally sequential events, such as initial peripheral blood vessel innervation followed by neuronal process innervation, regulated? Are molecular mechanisms of vascular and neuronal remodeling also shared
or codependent? How did the vascular system co-opt nervous system cues? What role do these common cues play in pathological angiogenesis or neurological disease? After more than a decade of applying insight from neuronal development paradigms to the vascular system, it is clear that this path of investigation is yielding key ndings. Much less is understood about the development, maturation, and maintenance of the blood-brain barrier (BBB). Understanding how the BBB develops and functions is of enormous importance. This need is fueled by the fact that the barrier hinders therapeutic treatment of neurological diseases and because it may also be at the center of neurodegenerative disease biology. Subsequent to the original discovery that the CNS microenvironment directs blood vessel differentiation to form the BBB (Stewart & Wiley 1981), the recent identication of the Wnt/beta-catenin signaling pathway regulating CNS angiogenesis and BBB formation is likely just the rst of many examples of regulatory cues derived from the CNS that drive brain endothelial cell development and differentiation. A unique Frizzled receptor ligand, Norrin, has recently been described to act via Fzd4, LRP5, and a tetraspanin molecule, TSPAN12, to regulate retinal vascular development specically (Junge et al. 2009, Ye et al. 2009). In addition, Norrin, Fzd4, Lrp5, and Tspan12 knockout mice show a vascular hemorrhage phenotype in the retina, consistent with the disruption of the retinal-blood barrier. These data suggest that unique Wnt signaling pathways have evolved in a regional-specic fashion to coordinate endothelial cell differentiation and barrier formation. Beyond Wnt signaling in CNS angiogenesis and BBB formation, many questions remain about how the BBB forms, matures, and is maintained. The barrier also consists of other cell types, including pericytes, astrocytes, and neuronal components. How is this complex NVU formed? How is the barrier actively maintained in the adult animal? What are the roles of the various cellular components in the development, maturation, and maintenance
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of the BBB? Discovering the molecular components that address these broader questions will deepen our understanding of the BBB, thus enabling researchers to address unresolved issues of drug penetration into the CNS
and barrier disruption in neurological disease paradigms. Using recent history as an example, clues to crack the mystery of BBB formation may be derived from applying principles of neuronal development to the vascular system.
DISCLOSURE STATEMENT
The authors are employees of Genentech, Inc.
ACKNOWLEDGMENTS
We apologize to our colleagues whose research we could not cite owing to space limitations. We thank Allison Bruce for the generation of illustrations and thank Kimberly Scearce-Levie, Weilan Ye, Joy Yu, Morgan Sheng, and Marc Tessier-Lavigne for their helpful comments on the manuscript. LITERATURE CITED
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Annual Review of Neuroscience
Contents
Volume 33, 2010
Attention, Intention, and Priority in the Parietal Lobe James W. Bisley and Michael E. Goldberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 The Subplate and Early Cortical Circuits Patrick O. Kanold and Heiko J. Luhmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 23 Fly Motion Vision Alexander Borst, Juergen Haag, and Dierk F. Reiff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 49 Molecular Pathways of Frontotemporal Lobar Degeneration Kristel Sleegers, Marc Cruts, and Christine Van Broeckhoven p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 71 Error Correction, Sensory Prediction, and Adaptation in Motor Control Reza Shadmehr, Maurice A. Smith, and John W. Krakauer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 89 How Does Neuroscience Affect Our Conception of Volition? Adina L. Roskies p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 109 Watching Synaptogenesis in the Adult Brain Wolfgang Kelsch, Shuyin Sim, and Carlos Lois p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131 Neurological Channelopathies Dimitri M. Kullmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 151 Emotion, Cognition, and Mental State Representation in Amygdala and Prefrontal Cortex C. Daniel Salzman and Stefano Fusi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 173 Category Learning in the Brain Carol A. Seger and Earl K. Miller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 203 Molecular and Cellular Mechanisms of Learning Disabilities: A Focus on NF1 C. Shilyansky, Y.S. Lee, and A.J. Silva p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221 Wallerian Degeneration, WldS , and Nmnat Michael P. Coleman and Marc R. Freeman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 245
Neural Mechanisms for Interacting with a World Full of Action Choices Paul Cisek and John F. Kalaska p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 269 The Role of the Human Prefrontal Cortex in Social Cognition and Moral Judgment Chad E. Forbes and Jordan Grafman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 299 Sodium Channels in Normal and Pathological Pain Sulayman D. Dib-Hajj, Theodore R. Cummins, Joel A. Black, and Stephen G. Waxman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 325
Mechanisms of Synapse and Dendrite Maintenance and Their Disruption in Psychiatric and Neurodegenerative Disorders Yu-Chih Lin and Anthony J. Koleske p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 349 Connecting Vascular and Nervous System Development: Angiogenesis and the Blood-Brain Barrier Stephen J. Tam and Ryan J. Watts p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379 Motor Neuron Diversity in Development and Disease Kevin C. Kanning, Artem Kaplan, and Christopher E. Henderson p p p p p p p p p p p p p p p p p p p p p p 409 The Genomic, Biochemical, and Cellular Responses of the Retina in Inherited Photoreceptor Degenerations and Prospects for the Treatment of These Disorders Alexa N. Bramall, Alan F. Wright, Samuel G. Jacobson, and Roderick R. McInnes p p p 441 Genetics and Cell Biology of Building Specic Synaptic Connectivity Kang Shen and Peter Scheiffele p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 473 Indexes Cumulative Index of Contributing Authors, Volumes 2433 p p p p p p p p p p p p p p p p p p p p p p p p p p p 509 Cumulative Index of Chapter Titles, Volumes 2433 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 513 Errata An online log of corrections to Annual Review of Neuroscience articles may be found at http://neuro.annualreviews.org/
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ANNUAL REVIEWS

392 392 395

VEGF: vascular endothelial growth factor

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

Shared Anatomical Patterning

Nf Nervous system Circulatory system

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

The Axon Growth Cone and Endothelial Tip Cell

Axon growth cone

Endothelial tip cell

Attractive guidance cues

Filopodium Lamellipodium Endothelial stalk cells

Capillary lumen (blood)

Endothelial tip cell Repulsive guidance cues

SHARED MOLECULAR MECHANISMS Axon Guidance Molecules and Angiogenesis

Vascular guidance molecules

The Slits and Robos in Vascular Guidance

The Semaphorins/Plexins/Nrps in Vascular Guidance

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

The Netrin/Unc5/DCC Family in Vascular Guidance

The Ephrin/Eph Family in Vascular Guidance

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

AXON GUIDANCE MOLECULES AS TARGETS FOR CANCER

Molecular Properties of the Blood-Brain Barrier

THE INTERFACE BETWEEN THE VASCULAR AND NERVOUS SYSTEMS

BBB: tight junctions, transporters, and transcytosis

-GT P450related MAO

Barrier regulation: molecular exchange between blood and CNS

Insulin, LDL, ApoE, transferrin (Tf), leptin

P-gp MRPs Tight junctions

Endothelial cell Abluminal (brain)

Ultrastructural image of endothelial tight junctions at the BBB

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

The Neurovascular Unit

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

The neurovascular unit (NVU)

Astrocyte end-foot Interneuron

Capillary lumen (blood)

Astrocyte end-foot Astrocyte end-foot Endothelial cell

Collagen IV = basal lamina

Collagen IV = basal lamina GFAP = astrocyte

THE BBB AND NEURODEGENERATIVE DISEASE

CNS Angiogenesis and BBB Formation: A Role for Wnt Signaling

Embryo neural tube cross-section

Angiogenesis and BBB formation

Capillary lumen (blood)

Wnts gradient Endothelial tip cell attraction?

Tight junction formation

Neural tube cavity

Nonbrain endothelial cells

Brain endothelial cells

Inactive dishevelled Active GSK 3b

Activated, dishevelled Inactive GSK 3b Axin

Axin Unstable -catenin

Wnt-responsive genes off

Transcription of Wnt-responsive genes

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

CONCLUSIONS AND FUTURE DIRECTIONS

www.annualreviews.org Angiogenesis and the Blood-Brain Barrier

Annual Review of Neuroscience

Volume 33, 2010

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