[Current Topics in Developmental Biology] Volume 80 || Similarities Between Angiogenesis and Neural...

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Similarities Between Angiogenesis andNeural Development: What Small AnimalModels Can Tell Us

Serena Zacchigna,* , {,1 Carmen Ruiz de Almodovar,* , {,1 andPeter Carmeliet * , {

* Department of Transgene Technology and Gene Therapy, VIB, Leuven, Belgium{ Department of Transgene Technology and Gene Therapy, K.U. Leuven

Leuven, Belgium

I. I

1B

Curren

Copyr

ntroduction

II. S
mall Animal Models to Study Blood and Vessel Guidance
A
. C
oth

t Top

ight 2

aenorhabditis elegans (Nematode Worm)

B
. D . melanogaster (Fruit Fly)
C
. Z ebrafish
D
. X enopus
III. V
ascular and Neural Cell‐Fate Specification
IV. M
olecular Links Between Angiogenesis and Neurogenesis
V. S
imilarities in the Organization of Vascular and Neural Boundaries
VI. M
olecular Cues Involved in Nerve and Vessel Guidance
A
. A xon Growth Cones and Endothelial Tip Cells
B
. C ommon Signals for Axon and Blood Vessel Wiring
VII. P
erspectives
A
cknowledgments
R
eferences
During evolution vertebrates had to evolve in order to perform more and

more complex tasks. To achieve this goal, they developed specialized tissues: a

highly branched vascular system to ensure that all tissues receive adequate

blood supply, and an intricate nervous system in which nerves branch to

transmit electrical signals to peripheral organs. The development of both sys-

tems is tightly controlled by a series of developmental cues, which ensure the

accomplishment of a complex and highly stereotyped mature network. Vessels

and nerves use similar signals and principles to grow, diVerentiate, and navigatetoward their final targets. Both systems share several molecular pathways,

highlighting an important link between vascular biology and neuroscience.

Moreover, the vascular and the nervous system crosstalk and, when deregu-

lated, contribute to medically relevant diseases. This new phenomenon, named

theneurovascular link,promises toaccelerate thediscoveryofnewpathogenetic

authors contributed equally to this manuscript.

ics in Developmental Biology, Vol. 80 0070-2153/08 $35.00008, Elsevier Inc. All rights reserved. 1 DOI: 10.1016/S0070-2153(07)80001-9

2 Zacchigna et al.

insights and therapeutic strategies for the treatment of both vascular and

neurological diseases. To study the development of both systems, scientists

are taking advantage of the use of several vertebrate and invertebrate animal

models. In the first part of this chapter, wewill discuss themore commonly used

animal models; in the second part, the striking similarities occurring during the

development of the vascular and the neural systems will be revised.� 2008, Elsevier Inc.

I. Introduction

During the course of evolution, vertebrates have learned how to perform

more complex and sophisticated tasks—this challenge could only be met by

the coincident development of two intertwined anatomic systems, blood

vessels and nerves: the former providing nutrients and the latter transmitting

electrical signals required for coordination. The vital importance of these two

systems was already acclaimed two millennia ago, when two major schools of

thought in the ancient Greece debated about the relative role of the brain or

the heart as the central source of life. The first school, headed by Plato,

supported the concept that the brain harbored the soul, whereas Aristotle

and his followers, in the second school, considered the heart and blood

vessels of major importance for life. As Aristotle stated, ‘‘The blood vessel

system can be compared to those of watercourses in gardens: they start from

one source and branch oV into numerous channels, so as to carry a supply

to every part of the garden.’’ Nowadays, we realize that there is no reason to

consider blood vessels and nerves as antagonists, as the Greeks once thought.

Instead, they have been recognized to share much more in common than

originally anticipated, in terms of development, molecular mechanisms of

wiring, and pathogenesis of disease. Thanks to the use of a variety of animal

models we have started to dissect the molecular pathways underlying blood

vessel and neuronal circuitry formation. Surprisingly, we are discovering that

both blood vessels and nerves use similar signals to grow, diVerentiate, andnavigate toward their final targets. Moreover, the vascular and the nervous

systems have been shown to crosstalk with each other and, when deregulated,

contribute to medically relevant diseases. In this chapter, we will first provide

an overview of the animal models that have mainly contributed to our

current understanding of the neurovascular link. We will focus on

the advantages of using either invertebrate (such as worms and flies) or

vertebrate (like fish and frogs) models to study the development and function

of nerves and blood vessels. Then, we will focus our attention in the parallel-

ism between neurogenesis and vasculogenesis, as well as between vessel and

nerve growth and navigation. Finally, we will discuss our current knowledge

about the molecular players certainly or possibly involved in blood vessel and

axon guidance.

STANDARD TRANSGENIC APPROACH

Transgene DNA ismicroinjected intothe male pronucleusof a fertilized murineoocyte

Injected oocytes are transferredto a pseudopregnant recipientmouse

Offsprings are screenedfor the transgene byDNA analysis

GENE-TARGETED TRANSGENIC APPROACH

Isogenic transgene DNAis introduced into ES cells(e.g., by electroporation) Drug selection is used

and the surviving coloniesare screened for thetransgene

Characterized targetedcells are microinjectedinto 3.5-day mouseblastocyst Blastocysts are transferred to a

pseudopregnant recipient mouse

Chimeric offsprings are identifiedand mated to test for germ linetransmission of the transgene

wt LacZ

C

B

A

Figure 1 Transgenic mice technology. (A) The standard transgenic approach is based on the

microinjection of the transgene into the male pronucleus of a murine oocyte, which is then

transferred to a 0.5‐day‐pseudopregnant recipient mouse. OVspring are screened for the presence

of the transgene. (B) In the more recent gene‐targeted transgenic approach, isogenic DNA [to the

embryonic stem (ES) cells being targeted] containing the transgene is introduced into the ES cells,

for instance, by electroporation. Drug selection is used and surviving colonies are screened for

the presence of the transgene. Targeted ES cells are then injected into 3.5‐day mouse blastocysts

and transferred to 2.5‐day‐pseudopregnant recipient mice. The incorporation of targeted ES cells

into the oVspring is determined by coat colour—that is, chimeric mice are generated that display

coat color of bothmice, fromwhich either blastocysts or the ES cells were derived. Chimeric mice

1. Angiogenesis and Neurogenesis 3

4 Zacchigna et al.

II. Small Animal Models to Study Blood and Vessel Guidance

Many aspects of biology are similar inmost or all organisms, but it is frequently

more straightforward to study particular aspects of development in specific

organisms. Over the past century, the mouse has become the premier mamma-

lian model system for genetic research. Scientists working in a wide range of

biomedical fields have gravitated to the mouse because of its close genetic and

physiological similarities to humans, as well as the relative ease with which its

genome can be manipulated and analyzed. Mouse models currently used in

biomedical research include thousands of unique inbred strains and genetically

engineeredmutants. There aremice prone to develop diVerent kinds of cancers,diabetes, obesity, blindness, Amyotrophic Lateral Sclerosis (ALS), Hunting-

ton’s disease, anxiety, alcoholism, and drug addiction. Innovative genetic

technologies have led to the production of custom‐made mouse models for

the study of a wide array of both disease and developmental processes. Un-

doubtedly, among the most important advances has been the possibility to

create transgenic mice, in which a foreign gene is inserted into the animal’s

germ line (Fig. 1). Alternative approaches, relying on homologous recombina-

tion, have permitted the development of tools to ‘‘knock out’’ genes by disrupt-

ing existing genes, even tissue specifically, or to ‘‘knock in’’ genes by altering a

mouse gene in its natural location.However, these techniques,which accounted

for an enormous progress in our understanding of the molecules implicated in

embryonic development as well as in the pathogenesis of diVerent diseases, areextremely expensive, laborious, and time consuming. In contrast, smaller ani-

mal models, such as worms, flies, fishes, and frogs, oVer the advantage to studygene functionwith amuch smaller budget andmuch shorter time frame, even at

ahigh‐throughput scale.This is probably the reasonwhynonmammalian, small

animal models have been extensively used in developmental biology, quickly

providing useful information about gene function, and pioneering medical

research to define novel therapeutic entry points. Indeed, genetic studies are

easier in small animals andmuchmore complex inmammals.With the fact that

the genomes from many diVerent species are already sequenced, there is cur-

rently a general eVort to functionally link genes frommodel organisms to their

counterparts in humans, in order to expand their utility for understanding

biological processes and diseases. In this perspective, only after confirmation

of the importance of one or more candidate genes in the small animal models,

they will ultimately be evaluated in the mouse or other larger animals.

are mated to determine whether the targeted ES cells have contributed to the germ line. Germ

line oVspring are finally screened for the presence of the transgene and mated to establish the

transgenic line. (C) The expression of the LacZ reporter gene, whose protein product �‐galacto-sidase can be detected histochemically by X‐gal staining, is evident in the LacZ transgenic mouse

(right) but not in the wilt type (wt) mouse (left).


In the following section, we will discuss the major advantages of the most

popular small animal models used in the study of angiogenesis and neurobi-

ology, together with the techniques that can be more suitably applied to each

model. However, it is important to mention that reliance of one model can

result in several experimental limitations, and can even distort our view of the

problem. For instance, some molecular pathways simply do not exist in

smaller animals, and thus cannot be studied there. In addition, small organ-

isms usually do not recreate the entire pathophysiology of complex human

diseases. Therefore, it is critical that diVerent models are considered at the

same time, as none of them can address all issues. Indeed, these models are

complementary, and only an integrated view of the information obtained by

the use of diVerent animals, further supplemented by human genetics, is

likely necessary for a correct understanding of any biological process.

A. Caenorhabditis elegans (Nematode Worm)

In the second half of the twentieth century, Sydney Brenner adopted the nema-

tode C. elegans as a laboratory animal model with the specific purpose of

studying the genetics of development, its nervous system, and its behavior.

Today,C. elegans is used to study amuch larger variety of biological processes,

including apoptosis, cell signaling, cell cycle, cell polarity, gene regulation,

metabolism, ageing, and sex determination (Kaletta and Hengartner, 2006).

Several key discoveries for basic biology as well as for medically relevant areas

were first made in this tiny worm. For instance, in 1993, the first presenilin gene

wasdiscovered inC. elegans (SundaramandGreenwald, 1993), andonly2 years

later, mutations in the human presenilin‐1 gene were associatedwith early onsetof familial Alzheimer’s disease (Sherrington et al., 1995). Another example

refers to type 2 diabetes. In 1997, genetic studies inC. elegans identified negative

regulators of the insulin signaling pathway, among daf‐16 (theC. elegans ortho-logue of the forkhead transcription factorForkhead box 01 transcription factor

(FOXO) (Ogg et al., 1997)). Five years later, FOXO loss of function was found

to rescue the diabetic phenotype of insulin‐resistance mice (Nakae et al., 2002).

Finally, C. elegans is not only an established genetic model but can also be

exploited to investigate the underlying mechanisms of whole animal pharma-

cology. For instance, the antidepressant fluoxetine has been shown to increase

serotonergic signaling inC. elegansby inhibiting theorthologueof the serotonin

reuptake transporter (SERT) (Ranganathan et al., 2001). This has stimulated a

number of investigations to identify additional modes of action of antidepres-

sants and to further elucidate the molecular mechanisms of depression.

C. elegans has a number of features that make it particularly attractive for

several areas of research. First, it is easy to grow as, although it usually lives

in the soil and feeds on various bacteria, it can readily be raised in the

6 Zacchigna et al.

laboratory on a diet of Escherichia coli. Second, it reproduces rapidly

and prolifically, developing from an egg to an adult worm of 1.3 mm within

3 days. Third, because of its small size, several assays can be carried out in

microtiter plates. Fourth, C. elegans genome was sequenced completely at the

end of 1998. It has about a hundredmillion base pairs and is surprisingly similar

(40% homology) to that of humans, which renders it a particularly attractive

model in the study of human development and disease. Overall, genetic

research is quite straightforward. C. elegans has five pairs of autosomes and

one pair of sex chromosomes, whose ratios determine the sex: if the sixth

chromosome pair is XX, then C. elegans will be a hermaphrodite, while an

X0 combination in the sixth chromosome pair will produce a male (in nature,

hermaphrodites are themost common sex).Hermaphrodites can self‐fertilize ormate with males but cannot fertilize each other. In the laboratory, self‐fertilization of hermaphrodites or crossing with males can be manipulated to

produce progeny with the desired genotype. Finally, what is unique about the

worm is that it is transparent at all developmental stages, facilitating the

identification of anatomical aberrations (e.g., as a consequence of mutations),

simply by inspecting the living animal.

1. C. elegans and Its Nervous System

C. elegans displays an invariant lineage, with exactly 959 somatic cells, of

which we know both the cellular anatomy and, in many cases also, the

function (Fig. 2). Among these, there are only 302 neurons, which are

classified in 118 functional classes, according to their involvement in diVerentbehavioral aspects, including the sensation of mechanical, chemical, olfacto-

ry, and thermal stimuli; the movement pattern during mating; and, quite

surprisingly for such a scanty number of cells, diVerent kinds of associativelearning. From an anatomical point of view, C. elegans lacks of a vascular

system, therefore allowing the study of several developmental processes

independently of blood vessel growth. In contrast, it has a prominent ventral

and a minor dorsal nerve cord, running along its longitudinal axis. The

centerpiece of its nervous system is the circumpharyngeal nerve ring, sur-

rounded by six ganglia; this structure is sometimes referred as the ‘‘brain’’

of the worm. With a few exceptions, neurons in C. elegans have a simple uni‐or bipolar morphology, typical of invertebrate animals. As discussed later

more extensively, in order to acquire a functional neuronal network, specific

connections (synapses) need to be formed between the growth cone of nerve

cells and their target cells. For that, neurons send axons that are guided to

their final destination by a process called axon guidance. Finally, once the

axon reaches its final target, the diVerentiation of presynaptic terminals

occurs. Molecules involved in axon guidance and synapse formation remain

extremely interesting subjects of investigation. At the neuromuscular

Pharynx

Intestine

Proximal gonad

Uterus

Anus

54

Distal gonad1

1

2 3

Body wall muscle

Amphidnerve

Hypodermis

Labialnerve

Pharynx

2

Nerve

Ring

3

Lateralganglion

Dorsal ganglion

DNC

4

Pseudocoelom

Intestine

VNCProximalgonad

Distalgonad

5

RectumLumbar ganglion

Seam cell

Anal depressormuscle

Dorsorectal ganglion

Ventral ganglion

A

B

Figure 2 Anatomy of an adult C. elegans hermaphrodite. (A) DIC image of an adult hermaphro-

dite, left lateral side. Scale bar 0.1 mm. (B) Schematic drawing of anatomical structures, left lateral

side. Dotted lines and numbers mark the level of each section in the lower part of the figure.

(1) Section through anterior head. (2) Section through the middle of head. (3) Section through

posterior head. (4) Section through posterior body. DNC, dorsal nerve cord; VNC, ventral nerve

cord. (5) Section through tail, rectum area (adapted from Altun, Z. F. and Hall, D. H. 2005.

‘‘Handbook of C. elegans Anatomy.’’ In WormAtlas http://www.wormatlas.org/handbook/

contents.htm).


junction (NMJ), nematode muscle cells are unusual: instead of motoneuron

axons navigating to contact the muscle, the situation is turned upside down,

and in C. elegans, muscle cells send cellular processes (muscle arms) to

contact motoneurons.

http://www.wormatlas.org/handbook/contents.htm

http://www.wormatlas.org/handbook/contents.htm

8 Zacchigna et al.

Genetic studies in the worm, by forward genetics or other manipulation

techniques, have shed light on several aspects of the nervous system develop-

ment. Furthermore, a plethora of components of synaptic neurotransmission

have been identified in the worm and linked to specific behavioral functions,

with similar approaches currently only carried out in the fruit fly Drosophila

melanogaster, as discussed later. In contrast, only few studies in C. elegans

have been based on electrophysiological recordings, with a still poor under-

standing of the molecular details of neurotransmitter function at the level of

the synapsis.

Recently, the introduction of new techniques has rendered the worm even

more attractive for the neurobiology field. The discovery of RNA interfer-

ence (RNAi), first in C. elegans, currently allows genome‐wide screening for

genetic components of specific neural functions. Notably, the 2006 Nobel

Prize in Physiology or Medicine has been awarded to the two American

scientists, Andrew Fire and Craig Mello, who first published their discovery

of the RNA i mechani sm in 1998 (Fire et al ., 1998 ). Altho ugh the syst em is

not equally reliable for all neural genes (Tavernarakis et al., 2000), the

development of sensitized genetic backgrounds has significantly improved

the eYcacy of gene knockdown by simply feeding or injecting double‐stranded RNA (dsRNA) targeting individual worm genes. In mammalian

systems, only short 22‐nucleotide dsRNA molecules are used, in order to

avoid an interferon response or nonspecific inhibition of protein synthesis

through dsRNA‐dependent protein kinases (Elbashir et al., 2001; Yang et al.,

2001). However, since neither of those responses occurs in C. elegans, it is

possible to use long dsRNA, which will give rise to many diVerent siRNA

molecules and attack the target mRNA at several points, thus enormously

increasing the eYciency of RNAi. Of notice, RNAi can also be induced at

any time during the animal’s life cycle, therefore oVering the opportunity to

study gene function at all stages. Another advantage of the use of RNAi in

C. elegans is that RNAi‐induced phenotype can be maintained over several

generations, simply by continuously feeding the worm on bacteria producing

the relevant dsRNA. Thus, the ease of the system, together with the avail-

ability of the relevant genome data, has enabled a novel, high‐throughput,systematic reverse genetic approach, known as genome‐wide RNAi, based on

libraries of either in vitro synthesized dsRNA or bacteria that produce

dsRNA (Mello and Conte, 2004; Tabara et al., 1998).

Finally, the introduction of the green fluorescent protein (GFP), also first

in C. elegans (Chalfie et al., 1994), now allows us to follow neural develop-

ment, axon migration, and synaptogenesis in vivo simply by looking at cell

fluorescence. In this context, neuronal specific green fluorescent transgenic

worms are being used (Christensen et al., 2002), thus enabling a number of

screens that have established milestones in our understanding of neural

patterning (Seifert et al., 2006).


B. D. melanogaster (Fruit Fly)

The fruit fly D. melanogaster, a small insect 3‐mm long, has been used for

decades to elucidate various developmental processes through its powerful

genetics. Its importance for biological research and human health was recog-

nized by the award of the Nobel Prize in Physiology and Medicine to

E. Lewis, C. Nusslein‐Volhard, and E. Wieschaus in 1995. Part of the reason

why several scientists use this insect as a model for their research is historical

(so much is known about it that it is easy to handle and manipulate) and part

is practical: it is a small animal, with a short life cycle of about 2 weeks, and is

cheap and easy to keep in large numbers. The Drosophila egg is about half a

millimeter long. It takes 1 day for the embryo to develop and hatch into

a warm‐like larva. The larva eats and grows continuously, molting 1, 2, and

4 days after hatching (first, second, and third instars). After 2 additional

days, it molts one more time to form an immobile pupa. Over the next 4 days,

the body is entirely remodeled to give origin to the adult winged form, which

then hatches from the pupal case and is fertile in about 12 hours (this timing

refers to 25 �C; at 18 �C, development takes twice as long).

Mutant flies, with defects in any of several thousand genes, are available,

and the entire genome, containing �14,000 genes, has recently been

sequenced. Drosophila has four pairs of chromosomes: the X/Y sex chromo-

somes and the 2, 3, and 4 autosomes. The magic markers that first put

Drosophila in the spotlight are polytene chromosomes. During larval growth,

the number of cells is kept constant but gene expression increases. As a

consequence, cells get much bigger and each chromosome divides hundreds

of times, but all the strands stay attached to each other. The result is amassively

thick polytene chromosome, which can be easily seen under the microscope.

Even better, these chromosomes have a pattern of dark and light bands,

reminiscent of a bar code, which is unique for each chromosome section. As a

consequence, any large deletion of major rearrangement can be identified, and

by the use of nucleic acid probes, individual cloned genes can be placed on a

polytene map.

1. Drosophila and Its Use to Study the Nervous System

As happens for C. elegans,Drosophila also lacks of a proper vascular system.

It only has a primitive heart, and hemolymph circulates through tissue spaces

devoid of any endothelial lining (HoVmann, 1995). The analysis of mutants

and the possibility to conduct genetic screening for particular phenotypes

have been extensively applied to decipher the molecular mechanisms leading

to a functional neuronetwork inDrosophila. Moreover, the progressive devel-

opment of genetic and cell imaging techniques has allowed neurobiologists to

generalize the use of various neuronal models at diVerent developmental

10 Zacchigna et al.

stages. It also allowed the execution of single‐cell analysis, as well as single‐cellgenetic manipulations. Several neuronal models have been chosen for their

best adequacy to study specific steps in the formation of functional networks,

highlighting some constants in the programming of neuronal connectivity.

In this respect, to study axon guidance, Drosophila oVers one of the most

useful models: the checkpoint decision at the midline. As further described

below in more detail, the central nervous system (CNS) of bilateral animals is

physically divided by the midline, through which neurons have to establish

communications in order to ensure proper coordination between the two sides

of the brain. Indeed, specific neurons send their processes across the midline,

creating communication lines, displayed as commissures. Therefore, several

CNS neurons face two choices regarding their projections, remaining ipsilat-

eral, thus avoiding the midline, or projecting contralaterally and crossing

the midline. This is the reason why the midline, with its ‘‘yes or no’’ choice

is an ideal paradigm to study how neurons make a directionality decision at a

checkpoint. InDrosophila, the embryonic abdominal CNS is composed of six

repetitive identical segments (A2–A7), each of which in turn can be divided

into two mirroring hemisegments. Each abdominal hemisegment contains

342 neurons, including 34 motoneurons leaving the CNS to innervate

30 abdominal muscles (Landgraf et al., 1997; Schmid et al., 1999). Staining

of the whole embryonic neuropile reveals a ladderlike structure with longitu-

dinal fascicles connected by a pair of anterior and posterior commissures

in each segment (Seeger et al., 1993; Fig. 3). In this context, two main groups

B

VNC

B

A

Figure 3 CNS development in Drosophila embryo. (A) Ventral view of a whole Drosophila

embryo stained with the monoclonal antibody BP104, which binds to and marks axons of all

neurons in the CNS. Theoutlinesof thebrain (B)andtheventralnervecord(VNC)areshownin (B).


of mutations have been identified: mutants in which commissures appear

thickened by an excessive number of crossing fibers, and mutants with thinner

commissures due to a reduced amount of fibers crossing the midline. These

opposite phenotypes lead to amodel in which excess crossing is due to a lack of

repulsion, while reduced crossing depends on absence of attraction (Tessier‐Lavigne and Goodman, 1996). Several molecules that generate this disorgani-

zation of midline crossing have been identified so far, and most of them will be

described and discussed in the following sections. Here, we want to emphasize

how these studies highlighted the importance of midline glial cells as an orga-

nizing center of neuronal projections. In fact, as further discussed later, midline

glia are responsible for the secretionof themajor guidance cues for commissural

axons, including Netrin and Slit, which provide attractive and repulsive

signals by interacting with frazzled and Robo receptors, respectively (Battye

et al., 1999; Harris et al., 1996; Mitchell et al., 1996). With its stereotypical

organization, the ventral nerve cord of Drosophila has allowed single‐cellanalysis manipulation, mainly through the use of the Gal4/UAS system

(Brand and Perrimon, 1993). In this system, Gal4 drives the expression of a

specific transgene in a restricted subset of cell types. Alternatively, fluorescent

proteins can be expressed in a cell of interest, allowing its complete morpholog-

ical visualization in vivo. In this respect, the generation of transgenic flies

expressing fluorescent reporters in specific subset of neurons has been extremely

useful for the visualization of axon pathfinding in vivo (Murray et al., 1998;

Salvaterra andKitamoto, 2001). In addition, such approaches have shown that

axons of a specific neuron can respond in a diVerent manner to mutations of

robo (the receptor for the midline axon‐repellent molecule Slit in flies) or

frazzled (the Drosophila receptor for the midline axon‐attracting molecule

Netrin). More precisely, the anterior corner cell (aCC) motoneuron normally

displays an ipsilateral axon and two groups of dendrites, one that crosses the

midline and the other one that remains ipsilateral. In frazzled mutants, aCC

axonal projection remains unaVected, while the dendrites are not anymore able

to cross the midline, indicating a role for frazzled in midline dendritic crossing.

In robo mutants, both aCC axons and dendrites remain normal. The story

appears completely diVerent for another motoneuron (RP3), which normally

projects its axon contralaterally and has two dendrite groups, growing away on

each side of themidline. In this case, frazzledmutants prevent the axon to cross

the midline, whereas robo mutants have normal axons but dendrites fail to

escape the midline (Furrer et al., 2003; Wolf and Chiba, 2000). These results

show that neurons are able to subcellularly integrate divergent signals in axons

and dendrites and make Drosophila an invaluable tool to visualize and

genetically manipulate single cells, thus shedding the light on the subcellular

regulation involved in axon guidance at the midline.

12 Zacchigna et al.

Another research field that has extensively exploitedDrosophila as a model

organism is the study of synaptic contacts between neurons. In fact, the

precise description of single brain cells and synapses and their amenability

to genetic analysis has provided a useful platform to unravel the mechanisms

and principles of synapse formation, which find many counterparts in other

animals. In particular, a detailed description of synaptic development and

structure has been reported for the Drosophila NMJ, which is easily accessi-

ble to manipulation and visualization. Fly’s NMJs are established in the

periphery in predictable combinations between individual motoneurons

and muscles (Landgraf et al., 1997), in a stepwise process. First, during the

period of pathfinding, each motor axon grows to its appropriate exit point

from the CNS, and either pioneers or joins the correct nerve branch in order

to reach its target muscle (Prokop and Meinertzhagen, 2006). Second, a

precise number of synapses form at each contact site, acquiring an appropri-

ate neurotransmitter composition and spatial distribution. More precisely,

synapses start to assemble at 13 hours of embryonic development, with most

of the contacts reaching structural and functional maturity at the time of

hatching (Prokop, 1999). However, during larval stages, NMJs dramatically

increase in size, and an additional de novo formation of NMJs occurs during

metamorphosis at the pupal stage. At each phase of postembryonic develop-

ment, NMJs adopt a stereotypic morphology, defined by nerve entry points,

branching pattern, and terminal size (Johansen et al., 1989). While the

mechanisms controlling NMJ formation and diVerentiation during embry-

onic development still remain largely unknown, a huge amount of informa-

tion has been accumulated on larval NMJ, in which synapse assembly clearly

requires a coordinated regulation by pre‐ and postsynaptic cells (Ashley

et al., 2005; Paradis et al., 2001). In contrast to NMJs, synapse assembly at

photoreceptor contacts only occurs during pupal development, although

some plasticity persists throughout adult life. The Drosophila visual organ

represents another paradigm model system for the understanding of neuro-

nal connectivity. Every photoreceptor terminal has to establish about

50 presynaptic sites, each arranging in a tetrad (a constellation of 4 postsyn-

aptic sites facing a single presynaptic release site), with a predictable blend of

postsynaptic cells (L1, L2, L3, and amacrine cells). Conversely, each L1 or

L2 lamina cell has to connect with the six photoreceptor terminals of its

lamina module, in order to pool the information from all the photoreceptors

of the same field of view (Meinertzhagen, 2000). Hence, Drosophila repre-

sents an outstanding model to dissect the mechanisms ensuring the correct

number of synapses and their specificity in order to establish reproducible

and functional microcircuits in the lamina.

Although the anatomy of the fly nervous system, consisting of �100,000

neurons, diVers significantly from that of vertebrates, many fundamental

cellular and molecular features of neuronal development and patterning are


conserved between vertebrates and invertebrates. This conservation makes

Drosophila a powerful system for basic studies of neuronal development and

function and, more recently, also for studies of neuronal dysfunction. In fact,

despite the prevalence and the severity of Parkinson’s disease, Alzheimer’s

disease, and multiple sclerosis, little is known about their molecular etiology;

therefore, disease‐modifying therapies have been so far remained largely

elusive. Although recent linkage studies identified a few genes responsible

for rare, heritable forms of neurodegenerative disorders, we still know very

little about the biological functions of those genes and how their mutations

lead to neuronal death. The completion of both human and Drosophila

genome‐sequencing projects has also revealed that a large fraction of human

genes involved in neurodegenerative disorders have highly conserved counter-

parts inDrosophila. For instance, theDrosophila genome encodes homologues

of five of the six Parkinson‐related genes identified so far (Whitworth et al.,

2006). In this respect, an important advantage of using Drosophila to under-

stand human disease is the possibility to perform genome‐wide genetic screensfor mutations in other genes able to modulate the phenotype associated with a

certain disease model. The power of this approach is the potential to identify

genetic pathways that cause the disease, as well as those that can influence its

progression, without requiring a priori knowledge of the function of the disease

gene. In this way, the human counterparts of suppressors identified from

screens using Drosophila define potential targets for therapeutic interventions.

C. Zebrafish

The zebrafish (Danio rerio) is a small tropical freshwater fish, which lives in

rivers of northern India, northern Pakistan, Nepal, and Bhutan in South

Asia. It possesses a unique combination of features that make it particularly

well suited for experimental and genetic analyses of early vertebrate develop-

ment (Anderson and Ingham, 2003; Kimmel, 1989; Ny et al., 2006). Adult

zebrafish are only 3‐ to 4‐cm long, so large numbers can be maintained

relatively inexpensively in a small space (Fig. 4). Furthermore, zebrafish

reaches sexual maturity in about 3 months, and a pair of zebrafish can

generate hundreds of embryos every fewweeks, making it possible to generate

thousands of progeny from a single breeding pair of fishes. Zebrafish eggs are

externally fertilized, providing readily access to the developing embryos at all

stages of development (Fig. 4). The fertilized embryos develop rapidly

making it possible to observe the entire course of early development in a

short time. As zebrafish embryos are optically clear, it is possible to directly,

noninvasively observe the major parts of the neural and vascular systems

(Ny et al., 2006). For all these reasons, zebrafish has recently emerged as an

Figure 4 Zebrafish as a model to study angiogenesis and neurogenesis. Image of a typical adult

zebrafish (A). Morpholino injection into one cell stage zebrafish embryo (B). Confocal image of

the vasculature of a 48‐hpf Fli:GFP transgenic zebrafish (C). Confocal image of a transverse

section of a 48‐hpf Fli:GFP transgenic zebrafish. Note ISVs growing in close apposition to

somites and neural tube (D). Confocal image of a 48‐hpf zebrafish immunostained with anti‐acetylated tubulin to detect axons. Note motoneuron axons growing out of the spinal cord and

commissural axons crossing the midline (E). DA, dorsal aorta; PCV, posterior cardinal vein;

DLAV, dorsal longitudinal anastomic vessel; PAV, paracordal vessel; ISV, intersegmental

vessel; SOM, somite; SC, spinal cord; MNA, motoneuron axon; CA, commissural axons.

14 Zacchigna et al.

advantageousmodel organism for the study of the stereotypic and evolutionary

conserved development of blood vessels and nerves.

1. Zebrafish and Its Use to Study Vascular Development

The vasculature is often diYcult to visualize, manipulate, and analyze in

higher vertebrates, mainly because it is deeply dispersed within other, fairly

opaque tissues, and its function is essential early in development.


Intersegmental vessels (ISVs) in zebrafish embryos develop within the first

2 days of life. Pathfinding of these vessels is stereotyped and likely genetically

programmed by an interaction of attractive and repulsive cues. In control

embryos, ISVs sprout from the dorsal aorta and grow dorsally between the

somites and neural tube; eventually they elongate and fuse with vessels from

the adjacent segments to form the dorsolateral anastomotic vessel (DLAV)

(Lawson and Weinstein, 2002). Secondary sprouts then come out from the

posterior cardinal vein (PCV) and migrate dorsally up to the horizontal

myoseptum to form the parachordal vessel (PAV) (Lawson and Weinstein,

2002; Fig. 4).

As described above, the zebrafish embryos are optically transparent allow-

ing the visualization of the vascular system. In addition, fish embryos are

small enough that they can receive suYcient oxygen via passive diVusion to

develop normally for a few days in the absence of blood circulation, on

perturbation of angiogenic processes. This is a unique advantage since per-

turbation of angiogenic processes in mice leads to early embryonic lethality,

complicating or impeding phenotypic analysis. Moreover, zebrafish is easily

amenable to forward and large‐scale genetic analyses. In this respect, an

angiogenesis assay in the regenerating fin of adult zebrafish was used to

screen for antiangiogenic activity of chemical compounds (De Smet et al.,

2006). For the purpose of investigating the mechanisms of angiogenesis in

zebrafish, a variety of tools and methodologies have been recently developed,

thus enormously amplifying the intrinsic advantages of the fish model. These

include cell‐fate and lineage analysis techniques, microinjection of biologi-

cally active molecules, gene knockdown by injection of morpholino antisense

oligonucleotides, transgenic zebrafish lines expressing fluorescent proteins

under the control of vascular‐specific promoters such as the promoter for fli1

(Lawson and Weinstein, 2002) or flk1 (Jin et al., 2005; Fig. 4), as well as

advanced microscopy technologies such as confocal microangiography and

high‐resolution mutliphoton time‐lapse imaging (Lawson and Weinstein,

2002; Motoike et al., 2000; Weinstein et al., 1995). As further discussed

later, the latter technique has supplied our first glimpses of the highly dynamic,

growth cone‐like behavior of growing endothelial cells (ECs) in vivo (Lawson

and Weinstein, 2002).

2. Zebrafish and Its Use to Study the Development of the Nervous System

Parallel to its wide use in cardiovascular developmental biology, many zebra-

fish mutants have been characterized, which exhibit specific defects in axon

guidance and/or synaptogenesis, thus making the fish an excellent model in

neurobiology as well (Hutson and Chien, 2002). Indeed, its nervous system is

simple and well characterized (Beattie, 2000). Its optical transparency allows

to image cell movement in vivo, or to ablate specific cells in order to screen for

16 Zacchigna et al.

defects in development or behavior (Liu andFetcho, 1999;Myers et al., 1986).

In addition, cells or tissues can be easily transplanted to test the autonomy of

gene function (Fricke et al., 2001). Like ISVs, motoneuron axons in zebrafish

follow a highly stereotyped pattern to navigate to their final destination.

Therefore, their migration during development has been analyzed to identify

new axon guidance cues (Beattie et al., 2002). Transgenic zebrafish expressing

GFP under specific motoneuron promoters, such as islet1 (Higashijima et al.,

2000) or hb9 (Flanagan‐Steet et al., 2005), have been developed and used to

study motoneuron axonal growth and guidance in vivo. Furthermore, GFP

has been cloned under several zebrafish neuronal promoters to follow the

development of the nervous system in vivo (Park et al., 2004a; Yoshida and

Mishina, 2003). Finally, zebrafish has also been used as an animal model to

study commissural axon fasciculation and midline crossing at the hindbrain

(Marx et al., 2001).

Of notice, instead of characterizing neurons based on their gene expression,

large genetic screens can be successfully used to search for genes according to

their function in zebrafish (Driever et al., 1996; HaVter et al., 1996). An

interesting example refers to space cadet, a mutant with abnormal locomo-

tion, both in response to escape stimuli and during normal swimming

(Granato et al., 1996). In these mutants, some hindbrain commissural fibers

fail to make normal connections to Mauthner neurons, the largest neurons in

the fish hindbrain, which are an essential component of the escape response

circuit (Lorent et al., 2001). Interestingly, another mutant, deadly seven (des)/

Notch‐1a, which has five times the normal number of Mauthner neurons,

does not display any defect in escape response (Gray et al., 2001). Further

analysis of these mutant larvae revealed that all the excess Mauthner neurons

were functionally incorporated into the escape circuitry, but with compensa-

tory decrease in the number of collateral departing from each Mauthner

neuron, as well as in the overlap of target innervation (Liu et al., 2003).

These data show that, similar to humans, neuronal circuitries in the zebrafish

embryo display a remarkable degree of plasticity, and it is tempting to

speculate that such developmental plasticity has been the prerequisite for

the evolution of new and more specialized neural networks.

D. Xenopus

The fundamental work of Hans Spemann on the Organizer, which entitled

him for the 1935 Nobel Prize, marked the emergence of the amphibian as an

important model system for the study of development. In most laboratories

of the molecular era, amphibian embryology is taken as a synonym of the

study of the African clawed frog Xenopus. This anuran has actually served as

a treasure trove for biologists to plunder in search of novel developmental


regulators. For instance, the definition of the role of transforming growth

factor‐� (TGF‐�) in mesoderm induction, as well as the identification of

several patterning genes, such as noggin, cerebrus, or chordin, has been

accomplished in Xenopus (Callery, 2006). The most used frog so far is the

tetraploid X. laevis, although many groups are now also adopting its more

genetically amenable diploid relative, X. tropicalis (Khokha et al., 2002).

Indeed, while the slower development and larger embryos of X. laevis make

them preferable for experimental embryology, the shorter generation time of

X. tropicalis renders genetics more feasible in this species, and several

mutants have been already identified (Noramly et al., 2005). The recent

availability of X. tropicalis genome sequence will result in great use for

several aspects of developmental biology. First, the analysis of cis‐regulatorsequences can be identified in silico and subsequently tested in vivo for their

capacity to drive the expression of appropriate reporter genes in transgenic

frogs. Second, there will be a great advantage for loss‐of‐function studies, as

morpholinos targeting candidate genes are likely to work more eYciently in

X. tropicalis than in X. laevis, because of the allelic redundancy in the

tetraploid. Finally, the sequenced genome will allow the use of additional

techniques such as chromatin immunoprecipitation and microarray analysis.

1. Xenopus and Its Use to Study the Vascular System

Of notice, both Xenopus and zebrafish models have been elegantly exploited

to better understand several aspects of cardiovascular physiology. Indeed,

Xenopus has been used for the screening of chemical compounds for cardio-

vascular development (De Smet et al., 2006). In fact, for many years, the

mouse has been the preferred model because of the known genomic sequence

and the possibility to create knockout as well as transgenic animals. However,

genetic mutations linked to severe cardiovascular dysfunction usually die

very early, making functional analysis diYcult, if not impossible. In contrast,

as already discussed for zebrafish, also in Xenopus, mutations aVecting the

heart at early stages do not aVect survival because they are independent of

blood flow for oxygen delivery early on (Territo and Burggren, 1998). There

are additional advantages in the use of these small animal models for studies

of embryonic cardiovascular function and development. Measurements of

heart rate, oxygen consumption, blood pressure, and hematocrit have all

been performed in developing embryos of both species (Bagatto et al., 2001;

Fritsche et al., 2000; Jacob et al., 2002; Pelster and Burggren, 1996; Schwerte

and Fritsche, 2003). Most of the times, as the larvae of both animals are

transparent, these parameters can be monitored noninvasively, by means of a

variety of video techniques. While the cardiovascular system becomes func-

tional at�24 hours postfertilization (hpf) in zebrafish, in Xenopus this occurs

at NF stage 33/34 (�48 hpf).

18 Zacchigna et al.

In addition, to study vascular development in Xenopus, transgenic frogs

expressing fluorescent proteins under the specific vascular promoter Tie2

have been developed (Ross Breckenridge and Timothy Mohun, personal

communication). Moreover, the use of this small animal model has also

been proven as a powerful model to study lymphangiogenesis (Ny et al.,

2005).

2. The Use of Xenopus to Study the Development of the Nervous System

The fact that, in Xenopus, neural development occurs much earlier than

vascular development renders the frog particularly useful for the study and

the dissection of the molecules involved in the neurovascular link. In fact,

during Xenopus embryogenesis, there is an early window (12–48 hpf ) in

which the eVect of a candidate gene directly on the nervous system can be

studied independently from any possible vascular eVect. The embryonic

Xenopus spinal cord has been proven to be an excellent model to follow the

growth pattern of living neurons (Moon and Gomez, 2005; Shim et al., 2005).

In addition, as mentioned above, it provides the advantage to study the

possible role of diVerent angiogenic molecules in midline axon guidance

without any primary vascular defect: commissural axons start crossing the

midline at approximately stage 23 and are finished by stage 28, when the first

angioblasts start to assemble in order to form the vascular system. Moreover,

by the blastomere injection method, it is possible to knock down a gene

specifically at the dorsal part of the neural tube, thereby targeting specifically

commissural neurons (Robles and Gomez, 2006). A representative picture of

a neuronal staining of a spinal cord from a stage 28Xenopus embryo is shown

in Fig. 5.

An additional system that has been providing increasing insight on axonal

wiring is the amphibian visual system, which undergoes profound remodel-

ing during metamorphosis. In tadpoles, the two eyes are laterally placed, with

retinal cells projecting exclusively to the contralateral side of the brain and no

binocular overlap (Holt and Harris, 1983). During metamorphosis, as the

skull changes its shape, the eyes migrate frontally, leading to a substantial

degree of binocular overlap, which will be essential to the predatory lifestyle

of the adult frog (Grant and Keating, 1986). Concomitantly, a new pattern of

retinal projections has to be established, connecting each retina to ipsilateral

thalamic nuclei (Hoskins and Grobstein, 1985). The pattern of cell produc-

tion in the retina is also completely changed during metamorphosis. Whereas

in tadpoles retinal stem cells proliferate symmetrically, a sudden shift toward

asymmetrical growth occurs at the beginning of metamorphosis, with more

cells being added at the ventral and temporal margin than at the dorsal and

Figure 5 Xenopus as a model to study angiogenesis and neurogenesis. Image of aX. laevis egg at

one cell stage (A). Stage 26–28 of a X. laevis tadpole (B). Fluorescent image of an Flk1:GFP

transgenic tadpole at stage 46–47, as observed GFP is expressed throughout the whole vascular

system (C).Wholemount neurofilament staining of a dissected spinal cord froma stage 28 tadpole

showing commissural axons crossing the midline (D). Transverse section of a stage 28 tadpole

showing commissural neuron cell bodies and axons (neurofilament staining in red) crossing the

midline (E). Nuclear staining (DAPI) is in blue. DA, dorsal aorta; PCV, posterior cardinal vein;

DLAV, dorsal longitudinal anastomotic vessel; H, heart; SC, spinal cord; CN, commissural

neuron cell bodies; CA, commissural axons; NC, notochord.


nasal margin (Beach and Jacobson, 1979). This is attributed to an increased

number of progenitor cells in the ventral as compared to the dorsal margin,

and it has been proposed that the extensive proliferation in the ventral retina

serves the production of neurons for the new binocular visual field and

compensates for the change in eye position (Mann and Holt, 2001). This

model system has been of particular relevance to study the role of ehprin

(Eph) family molecules in axon guidance (Mann and Holt, 2001), as

discussed later in more detail.

20 Zacchigna et al.

III. Vascular and Neural Cell‐Fate Specification

In this part of the chapter, wewill focus on recent evidences demonstrating that

cell‐fate determination of vascular and neuronal precursors is regulated in part

by common signals and genetic pathways. We will also describe how vascular

and neuronal precursors influence the cell‐fate decisionmaking of one another.

The main types of vascular cells, ECs, arise from mesodermal angioblasts

or hemangioblasts (Vogeli et al., 2006). The fact that diVerent ECs arise fromdiVerent angioblasts (Rovainen, 1991) indicates that angioblasts might have

or acquire positional and temporal identity during development. One of the

most important EC‐fate determinations is the diVerentiation of ECs into

arterial or venous ECs. In this context, molecules and pathways known to

be crucial for neural cell‐fate specification have also been shown to participatein arteriovenous cell fate. Although blood pressure can influence this cell‐fatedecision, recent findings indicate that arteriovenous EC fate is specified before

the onset of circulation. An example of common mechanisms regulating

vascular and neural cell fate is shown in zebrafish embryos: during develop-

ment, angioblast precursors for the dorsal aorta and the PCV are mixed in the

lateral posterior mesoderm (Zhong et al., 2001); the decision of those angio-

blasts to form either the aorta or the vein is induced by signals released by the

ventral endoderm and the notochord (Fouquet et al., 1997; Sumoy et al.,

1997). Similarly, neural progenitors also receive signals from the notochord

and the endoderm in order to diVerentiate. Genetic studies in mice, zebrafish,

and Xenopus have started to define the transcriptional code that determines

EC fate (Brown et al., 2000; Liao et al., 2000; Mikkola and Orkin, 2002). As

in neuronal cell fate, this code involves basic Helix‐loop‐Helix (bHLH)

transcription factors (Carmeliet, 1999) and inhibitors of DNA biding and

inhibitors of differentiation (Id) repressors (Lyden et al., 1999).

During embryonic development, neurons and glial cells arise from neu-

roectodermal stem cells (NSCs) located in the neural tube. In order to diVer-entiate, NSCs have to pass through three main phases: (1) the decision to

commit to a neural cell phenotype, (2) the determination of positional identity

(anteroposterior and dorsolateral), and (3) the developmental decision to

diVerentiate (Osterfield et al., 2003; Panchision and McKay, 2002; Temple,

2001). Notch expression in the neural tube is responsible for maintaining the

NSC potential by upregulating Hes1/5 type repressors (Gaiano and Fishell,

2002; Hitoshi et al., 2002). Vascular endothelial growth factor (VEGF), the

key player of angiogenesis (Lawson and Weinstein, 2002), has been impli-

cated also in maintaining NSC potential. In the avascular chicken retina,

VEGF is secreted by postmitotic retinal neurons and, by binding to its

receptor Flk1 expressed in neuronal progenitor cells, influences cell prolifera-

tion and suppresses retinal ganglion diVerentiation (Hashimoto et al., 2006).


The activation of the mitogen‐activated protein kinase MEK–ERK pathway

by VEGF was shown to be responsible for the proliferation of retinal neuro-

nal precursors, while the induction of a Hes1 response was responsible for the

blockage of the diVerentiation of these cells to retinal ganglion cells (RGCs)

(Hashimoto et al., 2006). When pro‐proliferation signals are downregulated,

neural cells start to diVerentiate. The subsequent positional identity of the

diVerentiating neural cells is determined by gradients of the morphogens

fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and

sonic hedgehog (Shh), which are secreted by tissues adjacent to the neural

tube (Osterfield et al., 2003; Patten and Placzek, 2000; Temple, 2001). Further-

more, they induce distinct neuronal cell subtypes in a dose‐dependent manner

among the morphogen gradient.

Shh, by regulating the levels of VEGF, is also involved in vascular cell‐fatedetermination. This was shown by experiments performed in zebrafish

mutants of sonic you (the homologue of Shh in mammals), where the forma-

tion of the aorta was impaired (Brown et al., 2000; Chen et al., 1996). Shh

was shown to induce expression of VEGF in the adjacent somites, which in

turn drove the arterial diVerentiation of angioblasts (Lawson and Weinstein,

2002; Lawson et al., 2002). Parallel to its role in neuronal cell fate, Notch

signals may also regulate the decision of hemangioblasts to diVerentiate intoeither endothelial or hematopoietic cells. Furthermore, Notch signaling also

influences arterial EC‐fate specification by acting downstream of VEGF

(Lawson and Weinstein, 2002). When Notch signaling is knocked down in

zebrafish embryos, there is a loss of artery‐specific markers and an ectopic

vein marker‐gene expression in the dorsal aorta. Consistently, the ectopic

activation of Notch signaling represses venous cell fate (Lawson et al., 2001,

2003). Recent insights have shown that the orphan nuclear receptor, COUP‐TFII, has a critical role in repressing Notch signaling to maintain vein

identity. COUP‐TFII is expressed specifically in venous endothelium, and

its mutation leads to activation of arterial markers in veins (You et al., 2005).

The neurovascular link is further supported by the cell‐fate specification

of neural crest (NC) cells. NC cells segregate from the dorsal portion of the

neural tube and migrate as a pluripotent cell population to several regions in

the embryo. NC cell diVerentiation is induced by a combination of a medio-

lateral gradient of BMP and an anteroposterior gradient of Wnt, FGF,

and retinoic acid (Aybar and Mayor, 2002; Etchevers et al., 2002; Knecht

and Bronner‐Fraser, 2002). NC cells are able to diVerentiate to cells of the

peripheral nervous system, melanocytes, and mesectodermal derivatives like

the craniofacial cartilage and bone (Dupin et al., 2001; Knecht and Bronner‐Fraser, 2002). Interestingly, NC cells also diVerentiate to the smooth muscle

cells (SMCs) that cover the blood vessels of the pharyngeal arch arteries, and

vessels in the jaws and in the forebrain (Dupin et al., 2001; Etchevers et al.,

2002). Migration and diVerentiation of NC cells then depend on intrinsic

22 Zacchigna et al.

cascades of transcription factors, receptors, and ligands (MaschhoV and

Baldwin, 2000) such as TGF‐�1 which directs NC cells to an SMC fate

(Shah et al., 1996). Notch‐3 signaling is also crucial for maintaining SMC

fate and, when mutated, causes cerebral arteriopathy and stroke (Kalimo

et al., 2002). Besides the NC, SMCs in other regions of the organism arise

from mesodermal or epicardial‐derived cells (Carmeliet, 2000; Etchevers

et al., 2002; Mikkola and Orkin, 2002).

Examples of crosstalk between both systems are given by the fact that

VEGF, when released from Schwann cells, induces arterial specification of

vessels tracking alongside these nerves (Mukouyama et al., 2002) in the embry-

onic limb skin. The induction of nerve‐mediated arterial diVerentiation as well

as the patterning of blood vessels along peripheral nerves is beneficial for both

systems. On one side, apart from supplying the nerves with oxygen and nutri-

ents, arterial vessels express neurotrophic factors such as nerve growth factor

(NGF), neurotrophin‐3 (NT‐3), and brain derived neurotrophic factor

(BDNF), which might be important to maintain the survival of growing

axons before arriving at their final peripheral destination. On the other side,

nerves control vasoconstriction and dilation of their flanking arteries. In a

study, Mukouyama et al. (2005) used Cre‐specific mouse lines to demonstrate

that, through binding to Np‐1, VEGF derived from motoneurons, sensory

neurons, and Schwann cells is required for in vivo arterial diVerentiation.Another example of crosstalk is the induction of specific blood–brain barrier

(BBB) ECs by glia‐derived neurotrophic factor (GDNF) and other glial cell‐derived factors (Carmeliet, 2003; Orte et al., 1999). TheBBB is a nonfenestrated

EC barrier where tight junctions seal oV the vascular lumen. It can be consid-

ered as a functional neurovascular unit, constructed by ECs, astrocytes, and

neurons, where mutual interactions between each component contribute to the

formation, maintenance, and function of the BBB (Kim et al., 2006).

IV. Molecular Links Between Angiogenesis and Neurogenesis

We will now discuss the direct link between angiogenesis and neurogenesis.

ECs have other functions than constituting pipelines for the supply of

oxygen. Instead, they are now known to release inductive cues for organo-

genesis and morphogenesis of various organs during development (Cleaver

and Melton, 2003; Compernolle et al., 2002; Eremina et al., 2003; Gerber

et al., 1999), as well as for neurogenesis and neural cell fate. ECs are present

at similar sites as NSCs and astroglial cells, and interact with these cell types

in a temporospatial manner (Huxlin et al., 1992; Zerlin and Goldman, 1997).

In specific areas of the CNS in mammals, NSCs proliferate in small clusters

around dividing capillaries—termed the vascular niche (Palmer et al., 2000).

Furthermore, ECs release factors such as BMP‐2, BDNF, and FGF, which

BMP

Somite

Shh

VEGF

Notch

Angioblast

Axial vein

Dorsal aorta

NSC

Neural tube

N

Notch

Figure 6 Vascular and neural cell fate. Notch expression in the neural tube maintains the NSC

potential. When Notch is downregulated NSC start to diVerentiate, then, gradients of morphogens

suchas Shh,which is secreted by the notochord, andBMP, determine the ventral and dorsal identity

of neural progenitors cells and induce distinct neuronal cell subtypes in a dose‐dependent manner

along themorphogengradient. Shhalso induces the releaseofVEGFfromthe somites,which in turn

acts on angioblasts to induce arterial and venous endothelial cell fate.


induce the diVerentiation of astrocyte precursors or NSCs (Mi et al., 2001;

Fig. 6). Additional evidence for a crosstalk between neural and vascular cells

is supported by the fact that VEGF, and semaphorin‐3A (Sema‐3A) antago-

nistically aVect neural progenitor cells (Bagnard et al., 2001) and ECs (Miao

et al., 1999). The link between the development of nerves and blood vessels is

also strengthened by the observation that conditions that increase neural

activity and stimulate neurogenesis also trigger angiogenesis (Kokaia and

Lindvall, 2003; Monje and Palmer, 2003).

New neurons are continuously being generated in the adult brain in

localized discrete regions such as the rostral subventricular zone (SVZ) and

the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). In these

two neurogenic areas of the adult brain, neural stem cells occupy niches

formed by both astrocytes and ECs (Lledo et al., 2006; Fig. 6). ECs are

critical components of these niches as they secrete soluble factors that main-

tain CNS stem cell self‐renewal and neurogenic potential (Shen et al., 2004).

When SVZ explants are cocultured with ECs, maturation, neurite out-

growth, and migration of neurons were enhanced (Leventhal et al., 1999),

indicating a role for ECs in neurogenesis. Furthermore, a recent report

24 Zacchigna et al.

showed that when coculturing NSCs with human ECs, a significant percent-

age of the NSC population converted to cells that did not express neuronal or

glial markers, but instead had a stable expression of multiple ECmarkers and

the capacity to form capillary networks (Wurmser et al., 2004). Recent

evidences show that VEGF is again the molecule that links angiogenesis

and neurogenesis. For example, in the adult songbird brain, neurogenesis

proceeds throughout life in the higher vocal center (HVC) of the neostriatum.

Using this animal model, Louissaint et al. (2002) could show that

testosterone‐induced angiogenesis as well as neurogenesis were associated

with increased VEGF expression within the HVC and BDNF production by

ECs. In addition, they showed that an inhibitor of VEGF receptor‐2(VEGFR‐2) blocked both the angiogenesis and the neurogenesis‐promoting

eVects of testosterone (Louissaint et al., 2002). Another illustration of the role

of VEGF in adult neurogenesis was shown in murine cerebrocortical cultures

as well as in the adult rat brain in vivo, VEGF stimulated proliferation of

neuronal stem cells in the SVZ and in the SGZ of the DG (Jin et al., 2002).

VEGF was also shown to induce neuroprotection, neurogenesis, and angio-

genesis after focal cerebral ischemia (Sun et al., 2003). Conversely, transient

forebrain ischemia‐induced cell proliferation and diVerentiation to mature

neurons in the hippocampal DG was shown to be attenuated when a VEGF

receptor tyrosine kinase inhibitor was administered intracerebroventricularly

after the induction of transient forebrain ischemia (Kawai et al., 2006).

Several evidences suggest that VEGF‐B, a VEGF homologue, could also

play a role in adult neurogenesis: (1) VEGF‐B is expressed in the brain, and

its expression is induced after brain injury (Nag et al., 2002); (2) VEGF‐Bknockout mice have been shown to exhibit increased infarct size and more

severe neurological deficits after stroke (Sun et al., 2004); and (3) VEGF‐Balso reduced hypoxia‐induced cell death of cultured cerebrocortical neurons

in vitro (Sun et al., 2004). It was indeed demonstrated that VEGF‐B stimu-

lates neurogenesis in the adult brain (Sun et al., 2006). Sun et al. showed that,

in VEGF‐B knockout mice, adult neurogenesis is reduced. Furthermore they

showed that the addition of VEGF‐B to neuronal cultures induced neuro-

genesis in vitro, and that intracerebroventricular administration of VEGF‐Bin rats promoted adult neurogenesis in the SGZ of the hippocampal DG and

in the forebrain SVZ (Sun et al., 2006).

V. Similarities in the Organization of Vascular andNeural Boundaries

The physical segregation of distinct diVerentiated cell populations is a

requirement for the organization of both the vascular and nervous network.

In both systems, similar families of molecules are responsible for the proper cell


separation. Accordingly, cells with common functions are attracted to each

other and cell boundaries are established between cells with diVerent properties.In that way, the identity of the many individual cell types in the complex

architecture is secured and organized. Members of the Eph family of receptor

tyrosine kinases and theirmembrane‐bound ephrin ligands (Davy and Soriano,

2005; Pasquale, 2005) have been shown to be the molecules responsible for

establishing these boundaries through bidirectional cell responses (Mellitzer

et al., 1999). Typical examples of boundary formation are the segmentation of

the vertebral hindbrain in rhombomeres in the nervous system (Cooke and

Moens, 2002; Krull, 2001; Tepass et al., 2002) and the separation between

arterial and venous ECs in the vascular system.

The hindbrain is transiently subdivided during development into repeated

segments called rhombomeres. Due to expression of ephrins and Eph receptors

in alternating segments (Cooke and Moens, 2002), rhombomeres are main-

tained as lineage‐restricted compartments. Ephrin–Eph signaling induces cell

repulsion between cells from diVerent rhombomeres, therefore maintaining

rhombomeres separated from each other (Cooke et al., 2001). In Xenopus and

zebrafish, it was shown that ephrin‐B signaling through Eph‐A4 is necessary

for rhombomere boundary formation (Cooke et al., 2005; Xu et al., 1995).

Furthermore, mutant zebrafish embryos that lack rhombomere boundaries,

due to a null mutation in the val gene, show a failure to establish

complementary expression pattern of Eph‐A4 and ephrin‐B2a between rhom-

bomeres. In addition, a publication showed that in Eph‐A4 mosaic zebrafish

embryos, Eph‐A4‐knockdown cells and Eph‐A4‐expressing cells segregate

from each other, suggesting that Eph‐A4 also regulate boundary formation

by promoting cell adhesion within cells of the same rhombomere (Cooke et al.,

2005). Taken everything together, Eph–ephrin interactions seem to contribute

to the sharpening of segments by regulating both repulsion at interfaces and cell

aYnity within rhombomeres.

Similar to the nervous system, ephrin–Eph signaling in the vascular system

establishes the boundaries between arterial and venous ECs (Adams and

Klein, 2000; Brantley et al., 2002; Torres‐Vazquez et al., 2003). Ephrin‐B2and Eph‐B4 are selectively and respectively expressed in arteries and veins of

mouse embryos, and this expression pattern seems to persist in the adult as

well (Gale et al., 2001; Shin et al., 2001). Ephrin‐B2‐null mice die at E10.5 as

a consequence of impaired vascular diVerentiation and arteriovenous remo-

deling, which results in a failure to form a properly branched capillary

network (Wang et al., 1998). Eph‐B4‐null mice essentially phenocopy

ephrin‐B2‐null mice, thus defining Eph‐B4/ephrin‐B2 as principal regulators

of vascular morphogenesis (Gerety et al., 1999). In a study with patients

suVering from venous malformations, it was found that while in normal

subjects, ephrin‐B2 and Eph‐B2 expression was restricted to arterial ECs,

in these patients, both molecules were found ectopically expressed in venous

26 Zacchigna et al.

ECs (Diehl et al., 2005). Parallel to what was described for Eph‐A4 as a

promoter of cell adhesion between cells within the same rhombomere, in the

vascular system, apart from a repulsive role for ephrin‐B2/Eph‐B4 signaling

in demarcating arteriovenous cell boundaries, ephrin signaling also regulates

the coherence of vascular cell subtypes of the same class. For instance,

conditional loss of ephrin‐B2 in vascular SMCs causes the mural vascular

smooth muscle coat to loosen up and mural cells to detach from each other

(Foo et al., 2006). Finally, ephrin‐B2 also controls interactions between

mural cells as well as between pericytes and the endothelium (Foo et al.,

2006). These interactions may reduce mural cell migration to ensure a proper

cover of mature vascular beds.

VI. Molecular Cues Involved in Nerve and Vessel Guidance

Understanding how axons can navigate throughout the body, eventually

reaching their final target, is one of the most interesting topics in the neuro-

biology field today. As they move, growth cones encounter and respond

diVerently to a complex array of attractive and repulsive signals at diVerentpoints along their pathway. Several families of axon guidance signals, along

with their cognate receptors, have been described so far. In this section, we

will present these molecules and the way by which their role in axon guidance

has been discovered. However, even if these ligand–receptor pairs have been

the stars of the show, there are probably more molecules in this ‘‘theater

troupe.’’ A main challenge for the future will be to understand how these

secondary players can help and cooperate to finely modulate the axon

guidance process.

A. Axon Growth Cones and Endothelial Tip Cells

Andreas Vesalius illustrated already five centuries ago that, although func-

tionally distinct, the vascular and the nervous systems are architecturally

similar and are structured into a ramifying and a hierarchically ordered

network. Both systems are composed of largely separate eVerent and aVerentnetworks (i.e., motor and sensory nerves in the nervous system, and arteries

and veins in the vasculature) (Carmeliet and Tessier‐Lavigne, 2005). Only

today, scientific evidence is emerging that vessels, which arose later in evolu-

tion than nerves, co‐opted several organizational principles and molecular

mechanisms that evolved to wire up the nervous system.

Apart from the similarities already described for neural and EC cell

fate, neuro‐ and angiogenesis, and tissue boundary formation, we can also

find striking similarities in the way axons and ECs find their way to their final

A

B C

Axonal growth cone

Endothelial tip cell

Ephrin

Npn/Plexin

Npn/Plexin

UNC5/DCC

UNC5

Robo

Slit

Robo

Netrins

Semaphorins

Eph

Ephrin

Ephrin

Eph

Eph

Figure 7 Morphological and molecular similarities between axonal growth cones and endothe-

lial tip cells. (A) The guidance of both axons (gray) and endothelial tip cells (green) is directed by

four major classes of ligands and their cognate receptors. (B) Scanning electron micrograph of an

axonal growth cone, terminating with numerous filopodial extensions (reproduced fromWessells

and Nuttall, 1978). (C)Multiphoton imaging of tip cell filopodia extending from the dorsal aorta

in a 22‐hour‐old zebrafish embryo.


destination. A highly motile structure located at the tip of the axon, the growth

cone, is the key player in axon pathfinding (Chilton, 2006; Fig. 7). By extending

filopodia and lamellipodia, the growth cone senses the microenvironment and

subsequently responds to a variety of guidance cues. Like that, the growth cone

reassesses its spatial environment and accurately selects a correct trajectory

among the maze of possible routes (Wen and Zheng, 2006).

In the vascular system, an emerging vessel is composed of specialized endo-

thelial ‘‘tip’’ cells present at the forefront and stalk cells located behind

(Gerhardt et al., 2003; Fig. 7). Notch signaling has been shown to participate

in the specification of endothelial tip cells at the forefront of the vascular

28 Zacchigna et al.

sprouts versus endothelial stalk cells trailing these tip cells in the nascent vessels

(Sainson et al., 2005). Endothelial tip cells share many similarities with axonal

growth cones, and also explore their environment by extending and retracting

numerous filopodia in saltatory fashion, suggesting that they direct the exten-

sion of vessel sprouts (Gerhardt et al., 2003). Endothelial tip cells, which

proliferate minimally, ‘‘pave the path’’ for the subjacent ‘‘stalk’’ ECs of the

growing vessel. In contrast, stalk cells proliferate extensively while migrating in

the wake of the tip cell, thus permitting extension of the nascent vessel.

B. Common Signals for Axon and Blood Vessel Wiring

Axons and vessels migrate to their final destination in a highly stereotyped

pattern. The long trajectory that growing axons follow to reach their final

target is split into multiple checkpoints that divide the path into a series of

shorter decision‐making events (Autiero et al., 2005). Like that, axons simplify

their task and navigate from one ‘‘intermediate target’’ or ‘‘choice point’’ to

the other (Chilton, 2006). They are usually attracted to a choice point by long‐range attracting signal produced by the intermediate target; once there, they

are expelled by short‐ or long‐range repellents also produced by cells at the

choice point (Chilton, 2006), in order to continue their trajectory. Like axons,

ECs have to migrate over short distances (similar to the short segments in

axons) to reach their final destination. Findings show that molecules that were

originally identified as axon guidance cues also play a role in blood vessel

guidance (Carmeliet and Tessier‐Lavigne, 2005; Fig. 8). Furthermore, axons

and vessels often take advantage of one another to follow the same path. In

some cases, vessels produce signals (such as artemin and NT‐3) that attractaxons to track alongside the pioneer vessel (Honma et al., 2002; Kuruvilla

et al., 2004). Conversely, nerves may also produce signals such as VEGF to

guide blood vessels (Mukouyama et al., 2002).

Four families of axon guidance cues, acting over a short range (cell‐ ormatrix‐associated signals) or long range (secreted diVusible signals), were iden-tified in the 1990s by genetic, biochemical, andmolecular studies (Fig. 8). These

families are Netrins and their deleted colorectal cancer (DCC) and uncoordi-

nated 5 (Unc‐5) receptors, Semas and their neuropilin (Npn) and plexin recep-

tors, Slits and their Robo receptors, and ephrins and their Eph receptors

(reviewed in Carmeliet, 2003; Chilton, 2006; Dickson, 2002; Huber et al.,

2003). Additionally, over the last few years, members from at least three

families of morphogens, previously described for their role in controlling cell

fate and tissue patterning, have been shown to act also as guidance cues: the

wingless/Wnt, hedgehog (Hh), and the decapentaplegic (Dpp)/BMP/TGF‐�families. The identification of these families of guidance cues has greatly

improved our understanding on the mechanisms of axon guidance and on the

1. Initial growth away from the roof plate: BMP-7/GDF-7; CS-PG

2. Projection along the lateral side of the spinal cord: TAG-1

3. Ventral and medial extension toward the floor plate:Shh/BOC; Netrin-1/DCC; Rig-1/Robo-3

4. Midline crossing toward the controlateral floor plate border:

5. Anterior turn to rostral projection: Wnt4/fz3

Slits/Robos; Sema-3B/Npn-2; Eph-B/ephrin-B; NrCAM; F-spondin

Shh

BMP-7rp

fp

1

2

3

45

Figure 8 Major guidance cues acting at the developing spinal cord midline. In the upper part,

the drawing on a transverse section of the spinal cord from a mouse embryo shows the pathway

of commissural axons, divided in five major segments, indicated by numbers. BMP-7 and Shh,

produced by the roof plate (rp) and by the floor plate (fp), respectively, create opposite morpho-

gen gradients, which contribute to commisural axon pathfinding in the developing spinal cord.

On the bottom, a list of the known molecules that have been implicated in the diVerent phases of

commissural axon guidance, as indicated by the numbers above.


wiring of the nervous system. However, many guidance events still remain

weakly understood and the number of guidance molecules identified seems

small, relative to the complexity of the nervous system. In the next paragraphs,

we will describe the role of these cues in the process of axon guidance and their

newly identified role as blood vessel guidance molecules.

1. Morphogens

Morphogens are signaling molecules that are produced locally, yet act at a

distance to pattern the surrounding field of cells in a concentration‐dependent manner. Members of the Wnt, TGF‐�, Hh, and more recently

30 Zacchigna et al.

FGF families have been shown to act as morphogens in both vertebrate and

invertebrate model organisms. The morphogen concentration gradient usually

translates into an activity gradient, which results in the diVerential activation oftarget genes in diVerent cells as a function of their distance from the morphogen

source. In this way, the so‐called ‘‘high‐threshold targets’’ respond only to high

levels of morphogen signaling, while ‘‘low‐threshold targets’’ respond to low

levels. During vertebrate CNS development, the paradigmatic example is

provided by the graded function of Wnt in patterning the neural plate along

its anteroposterior axis, and the antagonistic role of BMPs and Shh to specify

neuronal identity along the dorsoventral axis of the entire neural tube

(Bovolenta, 2005; Wilson and Houart, 2004). Interestingly, studies implicating

Shh, BMP, and Wnt in the control of growth cone movement have been

pushing the idea that morphogens can be reused later in development as

axon guidance cues (Butler and Dodd, 2003; Charron et al., 2003;

Lyuksyutova et al., 2003; Trousse et al., 2001; Wilson and Houart, 2004).

However, the first evidence that morphogenetic signaling further contributes

to growth cone steering originated from studies on FGF signaling on target

recognition.

a. Fibroblast Growth Factor. FGF proteins constitute a large family of

secreted factors composed of at least 23members, which give rise to a series of

splice variants and bind to cell surface tyrosine kinase receptors, encoded by

four genes (FGFR1‐4). Several FGFs and at least three receptors are

expressed in the developing CNS. For instance, FGFR1 is already expressed

in the cell body and growth cone of RGCs from the earliest developmental

stages (Brittis et al., 1996). When leaving the eye, RGC axons form the optic

nerve and reach the midline at the level of the optic chiasm, where in animals

with binocular vision, they face the choice to project contralaterally or stay

ipsilaterally. Extending through the optic tract, they finally establish synaptic

contacts with neurons in the optic tectum or superior colliculus (inmammals).

FGF signaling contributes to the proper RGC growth cone navigation in

diVerent segments of this pathway, as proven by the defasciculation and

growth cone dispersion observed in rats treated with FGFR‐blocking anti-

bodies (Brittis et al., 1996). In accordance, FGF8 and FGF19 have been

shown to be expressed by the optic fissure exactly at the time when RGC

axons are navigating through this region (Kurose et al., 2004). An analogue

mechanism seems operational in frogs, at least in the final steps of the RGC

trajectory. In fact, addition of FGF stimulated neurite extension from

cultured retinal neurons and induced aberrant axonal overgrowth in exposed

brain preparations ofXenopus embryos (McFarlane et al., 1995). Evidence of

a direct eVect of FGFs onRGC axons come from in vitro studies showing that

RGC growth cones are repelled by high levels of FGF2 (Webber et al., 2003).

Similar changes have been described for cortical neurons that, in response to


FGF2, increased the size of their growth cone, slowed their migration, and

increased axon branching (Szebenyi et al., 2001). Finally a peculiar role of

FGF in the regulation of axonal growth has been shown during trochlear

motoneuron pathfinding (Irving et al., 2002). Trochlear motoneurons, which

innervate eye muscles, send axons that project toward and extend within

the isthmus at the midbrain–hindbrain boundary, where Fgf8 expression is

required for a correct morphogenetic patterning in all vertebrates (Martinez

et al., 2001). Notably, FGF8 is later used to guide trochlear axons out of the

neural tube to form the fourth cranial nerve, as proven by the observation that

an antiserum against FGF8 or FGFR blockers causes axon misguidance and

defasciculation (Irving et al., 2002).

b. Sonic Hedgehog. Another morphogen involved in midline crossing of

diVerent types of axons is Shh. As described later in more detail, the major

chemoattractant for commissural axons at the ventral midline of spinal cord

is Netrin‐1. Indeed, in mice mutants for Netrin‐1 or its receptor DCC, many

commissural axon trajectories are foreshortened and misguided, failing to

invade the ventral spinal cord (Fazeli et al., 1997; Serafini et al., 1996).

However, some of them still reach the midline, indicating that other guidance

cues cooperate with Netrin‐1 to guide these axons. Further analysis of

Netrin‐1 knockout mice suggested that the floor plate produces additional

diVusible attractants for commissural axons. Given its expression by the

floor plate and its long‐range eVects already established in the spinal cord,

Shh was a candidate for a midline‐derived axonal guidance cue and it was

indeed shown to mimic the Netrin‐1 axonal chemoattractant activity of the

floor plate in vitro and in vivo (Charron et al., 2003). A putative receptor for

Shh, named biregional Cdon‐binding protein (Boc), has been identified on

commissural axons (Okada et al., 2006). Shh has also been proposed to act

as a negative regulator of RGC axon growth (Torres et al., 1996). Inactiva-

tion of the murine Pax2 gene alters the development of the optic chiasm in a

way that RGC axons never cross the midline. Notably, while Shh is down-

regulated in the chiasm during RGC axon migration in wild‐type mice, Shh

expression is maintained in the Pax2‐mutant mice (Torres et al., 1996),

suggesting that the continuous expression of Shh in the midline region

might impair RGCaxon growth, preventing them from crossing themidline.

In accordance, the ectopic expression of Shh at the midline is able to prevent

RGC axons from crossing the midline, without aVecting patterning and

neural diVerentiation in the eye (Trousse et al., 2001). The apparent contra-

diction between the eVects of Shh on commissural and retinal axons (attrac-

tion vs repulsion, respectively) may be related to the involvement of distinct

signaling pathways, resulting in opposite guidance eVects, as we will furtherdiscuss Netrins in the following paragraphs.

32 Zacchigna et al.

c. Bone Morphogenetic Proteins. An additional intriguing observation in

theNetrin‐1 andDCCmutants was that commissural axons initiallymigrated

ventrally for one‐third of their normal trajectory before becoming misrouted

(Fazeli et al., 1997; Serafini et al., 1996), indicating that additional cues might

guide their migration in the dorsal part. The proximity of commissural

neurons to the roof plate suggested that the roof plate itself might repel

commissural axons. Among a battery of candidate molecules, BMP‐7and BMP‐6, both expressed by the roof plate, can indeed mimic the chemor-

epellent activity of the roof plate in vitro (Augsburger et al., 1999). Further

genetic studies indicated that the roof plate BMP‐related chemorepellent

activity, which guides the initial trajectory of commissural axons in the

developing spinal cord, consists of a BMP‐7 and growth diVerentiationfactor‐7 (GDF‐7) heterodimer (Charron and Tessier‐Lavigne, 2005). BMPs

and the related GDFs are signaling molecules of the TGF‐� superfamily,

required for the specification of the spinal cord dorsal interneurons, including

commissural neurons, the choroid plexus in the forebrain, and granule cells in

the cerebellum (Alder et al., 1999; Hebert et al., 2002; Lee et al., 1998). Thus,

gradients of BMPs ad Shh appear to cooperate at least twice during the

development of the neural tube: first in the specification of cell fate, and

later to guide commissural axons to the ventral midline. Whereas a single

Shh molecule seems to play both roles, it remains to be determined whether

the same BMP family members can accomplish both functions or, instead,

whether diVerent BMP molecules independently perform each role.

d. Wnt. The last class of morphogens implicated in axon guidance is the

Wnt family of proteins, which are secreted molecules implicated in tissue

patterning, as well as in cell proliferation and diVerentiation in a variety of

tissues. By binding to the frizzled (Fz) receptors, Wnt activates several signal-

ing pathways, which ultimately result in changes in both gene expression and

cell adhesion. The property of Wnt proteins to rearrange the cytoskeleton

during axonal growth cone extension suggested that they might also be

involved in axon guidance. Intriguing evidence of a guidance role for Wnt

protein was obtained by studying the nervous system development in

Drosophila, where commissural axons have to choose between projecting

either through the anterior or through the posterior commissure. A major

role of the derailed (Drl) receptor in this decision has been established, based

on the initial observation that its expression is restricted to the growth cones of

axons that project into the anterior commissure (Callahan et al., 1995).

Indeed, in the absence of Drl receptor, neurons that normally cross the

anterior commissure often project to the posterior commissure. Conversely,

ectopic expression of Drl receptor in posterior neurons forces them to project

in the anterior commissure (Bonkowsky et al., 1999). Thus,Drl receptor seems

to be necessary and suYcient to direct axons into the anterior commissure.


The fact that Drl receptor contains a Wnt inhibitory factor domain led to

propose a model in which a Wnt protein might act as a repellent at the

posterior commissure, forcing axons to project anteriorly by binding Drl

receptor. In accordance with this idea, loss of Wnt5 function resulted in

commissural axon defects similar to those observed in drl mutants, whereas

overexpression of Wnt5 throughout the midline prevented the formation of

the anterior commissure, but not in drl mutants. Subsequent biochemical

work demonstrated that Drl receptor actually functions as a receptor for

Wnt5 (Yoshikawa et al., 2003), providing the first evidence of a ligand for

the Drl family of receptors and suggesting that other members of the family

might also act as Wnt receptors.

Once at the contralateral side of the floor plate, commissural axons have to

make a sharp turn projecting in a rostral direction—which are the cues that

control this step? Wnt4, which is expressed in an increasing posterior to

anterior gradient, at least at the mRNA level, appears to be a main player

in this pathway. Ectopic expression of Wnt4 was found to redirect postcross-

ing axons in vitro, and soluble Wnt inhibitors induced postcrossing commis-

sural axons to stall and turn randomly along the anteroposterior axis

(Lyuksyutova et al., 2003). In addition to Wnt4, Shh has also been identified

as a major candidate to guide commissural axons in the rostral direction

along the longitudinal axis of the spinal cord in the chick embryo (Bourikas

et al., 2005). Indeed, inhibiting Shh activity by RNAi or blocking antibodies

led to axon stalling, with some axons turning caudally or rostrally, apparent-

ly in a randommanner. Finally, postcrossing commissural axons were shown

to avoid ectopic Shh in vivo (Bourikas et al., 2005), providing strong evidence

that Shh is essential for the normal guidance of commissural axons along the

longitudinal axis of the spinal cord. Although it is not yet known whether

Shh guides postcrossing commissural axons in rodents, nor whether Wnt4

guides postcrossing commissural axons in chick, it seems particularly inter-

esting to note that complementary Wnt4 and Shh gradients might act in a

cooperative manner in the rostral guidance of commissural axons (Charron

and Tessier‐Lavigne, 2005).

2. Netrins and Their Unc‐5 and DCC Receptors

Netrins are a family of proteins highly conserved from C. elegans to mam-

mals. Inmammals, there are fourNetrin homologues (Netrin‐1, ‐2, ‐3, and ‐4).Their structures share similarities with the short arms of laminin‐� (Netrin‐1and ‐3) or �‐chains (Netrin‐4). They contain a laminin VI domain, three EGF‐like repeats, and a C‐terminal domain that can bind heparin, heparan sulfate

proteoglycans, or membrane glucolipids, thereby allowing interaction with

components of the extracellular matrix or the cell surface (Barallobre et al.,

2005). The extent of their diVusion is determined by both their expression level

34 Zacchigna et al.

and the concentration of binding sites in the surrounding tissue. Netrins bind

two families of receptors, each with a single transmembrane domain. In verte-

brates, the DCC receptor family comprises DCC and Neogenin, which share

homology to Unc‐40 in C. elegans and frazzled in Drosophila (Barallobre et al.,

2005). The other family of receptors that bind Netrins is the Unc‐5 family.

Vertebrates have four homologues, Unc‐5‐A, ‐B, ‐C, and ‐D, which are ortho-

logues of Unc‐5 inC. elegans (Ba ra ll ob re et al., 2005). Netrins have a dual role

in axon guidance; they can act either as attractant or as repellent molecules

(Barallobre et al., 2005). Signaling of Netrins through DCC receptors induces

axon attraction, while repulsion is generated by the binding of Netrins to Unc‐5receptors (short‐range repulsion) or to a combination of Unc‐5 and DCC

r ec ept or s ( long ‐range repulsion) (Hong et al., 1999; Keleman and Dickson,

2001).

As menti oned previou sly, in species with bilate ral symm etry, neuron s

con nect from one side of the CN S to the other by project ing axons across

the midl ine via co mmissure s. In this way, a pro per and coo rdinated functio n

of the brain is ensured. Netrin ‐ 1 is express ed in the floor plate and inneu roepitheli al cell s of the ventra l region of the spinal cord during develop-

ment ( Barallo bre et al ., 2005 ). The analys is of Netr in ‐ 1‐ deficient mice showe d

that commi ssural axons, whi ch express DC C on their surfa ce, althoug h they

star ted to grow toward the floor plate later on their trajector y they wer e

stalled or mis routed on their way to the midline (Se rafini et a l., 1996 ).

A simila r phen otype was found wi th DCC ( Fazeli et al ., 1997; Serafi ni

et al., 1996). In wild type embryos, once axons have reached the midline,

their response to the chemoattractant activity of Netrin‐1 is silenced to avoid

stalling at the midline. This is achieved by the commissureless (Comm)

recept or in Dros ophila and by Robo ‐ 3 in mou se (Section 3), whose express ion

levels become higher in postcrossing commissural axons, by forming a

complex with DCC that inactivates Netrin‐1 attractant activity (Stein

et al., 2001).

The analysis of developing ISVs in zebrafish embryos revealed an unex-

pected role for Unc‐5b and Netrin‐1a in vessel guidance. The initial sprouting

of the ISVs in zebrafish knockdown of either Unc‐5b or Netrin‐1a was

unaVected, but when they reached the level of either the horizontal myosep-

tum or the floor plate (which normally express Netrin‐1a), they deviated

laterally instead of extending dorsally (Lu et al., 2004). Furthermore, capil-

lary branching was increased, which together with the ISV defects resembles

the phenotype observed in Unc‐5b‐deficient mice (Lu et al., 2004). When

injecting recombinant Netrin‐1 into hindbrains of E10.5 wild‐type mouse

embryos, a marked retraction of the tip cell filopodia occurred, compared to

injected control. In addition, this eVect was abolished in Unc‐5b knockout

mice (Lu et al., 2004).These results suggest thatNetrin‐1,bybinding toUnc‐5B,inhibits vessel branching at specific ‘‘signaling points.’’ In contrast, other


studies proposed an attractive role for Netrin‐1. In contrast, using also

zebrafish as animal model, it was shown that, after knockdown of Netrin‐1a,the ISVs and DLAVs formed normally but the formation of the PAVs

was inhibited (Park et al., 2004b; Wilson et al., 2006). The authors proposed

that Netrin‐1a is required to induce EC migration alongside muscle pioneer

cells when forming the PAV (Park et al., 2004b; Wilson et al., 2006). In the

same study, Netrins also promoted neovascularization and reperfusion in a

murine model of peripheral vascular disease (hind limb ischemia) (Wilson

et al., 2006). Why has Netrin‐1 been described to have repulsive (Lu et al.,

2004) and attractive activities (Wilson et al., 2006) in blood vessel guidance?

One possible reconciling hypothesis is that depending on the receptor to

which it binds, Netrin‐1 may act as a repulsive or attractant cue for ECs.

3. Slits and Their Roundabout Receptors

Slits are proteins that are highly conserved from C. elegans to vertebrates

(Brose and Tessier‐Lavigne, 2000). They have multiple binding domains,

including four leucine‐rich repeats (LRRs), nine EGF‐like repeats (seven in

Drosophila), and a C‐terminal cystenin knot. In Drosophila, there is only

one Slit; in contrast in mammals, three family members have been identified

(Slit‐1, ‐2, and ‐3). Slits signal through binding single transmembrane recep-

tors of the roundabout or Robo family (Kidd et al., 1998). These receptors

contain an extracellular region with five immunoglobulin (Ig) domains and

three fibronection type III repeats. In Drosophila, Slit binds to one Robo

receptor; in vertebrates, four Robo receptors (Robo‐1, ‐2, ‐3, and ‐4) areknown, with Robo‐4 (also known as magic roundabout) being structurally

divergent from the other Robos.

Slits are expressed in the nervous system midline (Brose and Tessier‐Lavigne, 2000). They have been described to repel certain axons but, con-

versely, also stimulate branching and elongation of others (Kidd et al., 1999;

Li et al., 1999; Wang et al., 1999). Slit proteins have been shown to regulate

midline guidance in Drosophila and vertebrates. In flies, Slit is expressed at

the ventral midline, where it acts (through Robo) as a short‐range repellent toprevent ipsilateral axons from crossing the midline and commissural axons

from recrossing (Kidd et al., 1999). In flies lacking Slit, axons that normally

do not cross the midline do so, and axons that cross it only once can then

cross it several times.

Midline defects at the optic chiasm and in major forebrain tracts were also

observed in mice lacking Slit‐1 and Slit‐2, yet spinal commissural axons

appeared unaVected (Plump et al., 2002). The analysis of a triple mouse Slit

knockout showed that when all six Slit alleles are removed, commissural

axons reached the midline, but then many of them failed to leave it, while

others recross it (Long et al., 2004).

36 Zacchigna et al.

If commissural axons are attracted to the midline by Netrin‐1, how can

they cross it if they are also repelled by Slits? A highly controlled mechanism

guarantees that Slits expel crossing axons only after and not before they cross

the midline. Several mechanisms have been proposed to underlay this switch.

First, Robo receptors are expressed at low levels in precrossing commissural

axons (Kidd et al., 1998; Long et al., 2004). Second, Robo receptors are

expressed in precrossing commissural axons but are forming inactive com-

plexes (Sabatier et al., 2004). In both ways, precrossing commissural axons

are insensitive to Slit repulsion. In Drosophila, the regulatory protein Comm

keeps the Robo receptor intracellularly away from the plasma membrane,

thereby lowering Robo surface expression in precrossing commissural axons

(Keleman et al., 2002, 2005). Once commissural axons have crossed the

midline, Comm repression is lost and Robo becomes expressed at the axon

surface. Consequently, axons become sensitive to Slits and are expelled from

the midline. In mammals, Robo‐1 and Robo‐3 are expressed in precrossing

commissural axons. Robo‐3 functions as an ‘‘anti‐Robo’’ as it plays a similar

role as Comm inDrosophila: it silences Robo‐1 and blocks the binding of Slits

to Robo‐1, thereby eliminating its repulsive activity (Sabatier et al., 2004).

After crossing, Robo‐3 remains expressed but Robo‐1 and Robo‐2 become

upregulated (Sabatier et al., 2004). Like this, they overwhelm the negative

regulation of Robo‐3 and ensure that Slit‐mediated repulsion in axons starts

only after they have crossed the midline (Sabatier et al., 2004).

Robo‐4 was described as a vascular‐specific Robo homologue (Park et al.,

2003) which is selectively expressed in developing blood vessels during embry-

onic development (Park et al., 2003) and expressed only at sites of active

angiogenesis including tumor vessels during adulthood (Huminiecki et al.,

2002). A Robo‐4 knockdown study in zebrafish showed that some Robo‐4‐expressing ISVs failed to sprout from the dorsal aorta or arrested midway

through their dorsalmigration path, whereas others deviated from their normal

dorsal trajectory (Bedell et al., 2005). By in vitro gain of function approaches,

Kaur et al. (2006) showed that Robo‐4 activates Cdc42 and Rac Rho GTPases

in ECs. When they knocked down Robo‐4 in zebrafish, they could observe

lower amounts of active Cdc42 and Rac1 as well as a lack of direction of

isolated Robo‐4 knock down (KD) angioblasts (Kaur et al., 2006). Although

it is not clear what ligand is signaling throughRobo‐4 to create that phenotype,in vitro experiments showed that Robo‐4 on human ECs bound soluble Slit‐2and that this binding inhibited EC migration, suggesting a repulsive role for

Robo‐4/Slit‐2 in angiogenesis (Park et al., 2003).However, another study failed

to detect binding of Slit‐2 to Robo‐4 (Suchting et al., 2005).More evidences demonstrating a role for Slits and Robos in angio-

genesis come from in vitro studies in which the exposure of Robo‐1‐positivehuman umbilical vein ECs (HUVEC) to a Slit‐2 source stimulated their

chemotaxis. In vivo experiments also supported an attractive role for Slit‐2 on


Robo‐1‐expressing vessels (Wang et al., 2003). These results are thus in

disagreement with the documented repulsive activity of Slit‐2 when binding

to Robo‐4, as mentioned above (Park et al., 2003). Additional studies will help

to clarify the role of Slit‐2 in developmental and pathological angiogenesis and

explain whether Slit‐2 could have opposite eVects (repulsion or attraction)

depending on the Robo receptor subtype to which it binds.

4. Semas and Their Npn and Plexin Receptors

Todate,more than 20Semas have been identified and categorized, according to

sequence similarities and structural properties, into 8 classes. They belong to a

large family of both membrane and secreted proteins, characterized by the

presence of a highly conserved 500‐amino acid extracellular domain (Sema

domain), whichmediates the binding tomultimeric receptor complexes,mainly

composed of plexins and Npns, but often including additional molecules

(Suchting et al., 2006). Generally, membrane‐associated Semas bind directly

to plexin receptors, while class III secreted Semas (Sema‐3A‐F) require Npn

receptors, which seem not to signal themselves but act as coreceptors for plexin

signaling. Originally, genetic studies in Drosophila and mice implied Semas as

major cues in axon guidance and neuronal cell migration. In general, they are

considered to act as repellents, though Sema‐3A can also function as a chemo-

attractant, depending on the intracellular levels of cyclic nucleotides (Carmeliet

and Tessier‐Lavigne, 2005). The main receptors for Semas in the nervous

system are plexins, either alone or complexed with Npn receptors. As the

intracellular domain of Npns is extremely short, they associate with plexins in

order to induce an intracellular signaling mechanism. While Sema‐3A binds

only Npn‐1, other members of the family, such as Sema‐3B, Sema‐3C, andSema‐3F bind both Npn‐1 and Npn‐2 (Chen et al., 1998; Takahashi et al.,

1998).

Surprisingly, Npn‐1 and Npn‐2 were also found to be expressed by ECs

and to associate as coreceptors with VEGFR‐1 and ‐2. The expression of

Npn‐1 in ECs increases the aYnity of VEGF164 for VEGFR‐2, thus enhanc-ing VEGFR‐2 signaling, leading to EC chemotaxis and other angiogenic

steps (Miao et al., 1999). In contrast, when complexed with VEGFR‐1,Npn‐1 seems to prevent the binding of VEGF to this receptor (Fuh et al.,

2000), but the general relevance of this finding remains to be determined. The

heparin‐binding form of PlGF (PlGF‐2) and VEGF‐B, two additional mem-

bers of the VEGF family, also bind Npn‐1 (Makinen et al., 1999; Migdal

et al., 1998). Npn‐2 was shown to interact with VEGFR‐2 and ‐3 and act as a

coreceptor to enhance EC response to VEGF and VEGF‐C (Favier et al.,

2006). Therefore, Npns bind to two unrelated ligand families, the Sema

family and the VEGF family, which suggests the existence of common

molecular mechanisms in these two biological processes.

38 Zacchigna et al.

Sustaining such a neurovascular link is the fact that VEGF antagonizes the

proapoptotic and collapsing eVect of Sema‐3A on axons (Gu et al., 2002). On

the other hand, ECs respond to Sema‐3A by decreasing their migratory

capacity, as well as microvessels and lamellipodia formation, eVects can be

reversed by VEGF (Miao et al., 1999). Moreover, VEGF induces the prolif-

eration of diVerent tumor cell lines, while Sema‐3A exerts a proapoptotic

eVect on the same cells (Guttmann‐Raviv et al., 2006). The opposing eVect ofVEGF and Sema‐3A suggests that (1) they compete for overlapping binding

sites in the extracellular domain of a series of shared receptors or (2) they

provide independent, opposite intracellular signals to their target cells. Using

transgenic mice which express a mutant Npn‐1 that is unable to bind to

Sema‐3A, but still able to bind VEGF (npn‐1Sema� mice), it was shown that

neural morphogenesis was severely aVected without any eVect in the vascular

system, indicating that Sema‐3A/Npn‐1 is dispensable for vascular develop-

ment (Gu et al., 2003). Moreover, vascular malformations were caused by

conditional silencing of Npn‐1 in ECs presumably because of an impaired

VEGF/Npn‐1 signaling. A role for Sema‐3C in the vascular system has

also been described (Banu et al., 2006). Sema‐3C was shown to regulate

glomerular EC function by stimulating integrin phosphorylation and

VEGF120 secretion (Banu et al., 2006).

ThereceptorPlexin‐B1,which iswidelyexpressedinthenervoustissues, isalsoexpressed in adult ECs (Basile et al., 2004). The signaling Sema‐4D/Plexin‐B1induces repulsion in developing axons and maintenance of established neural

pathways in the adult (Kruger et al., 2005). In contrast, the binding of Sema‐4Dto Plexin‐B1 induces tubulogenesis and migration of ECs, and angio-

genesis in vivo (Basile et al., 2004). Furthermore, Sema‐4D was shown to be

expressed in head and neck squamous cell carcinoma (HNSCCs) and to stimu-

late tumor angiogenesis (Basile et al., 2006). Results obtained in zebrafish and

mouse embryos confirmed that Plexin‐D1 is involved in vessel morphogenesis

(Weinstein, 2005). Plexin‐D1 is expressed in ECs in zebrafish embryos; comple-

mentary, class III Semas are expressed in somites (Weinstein, 2005).

These Semas, by binding to Plexin‐D1, act as a repulsive cue for ECs, permit-

ting them to select the appropriate ISV branching site (Weinstein, 2005). Inter-

estingly, Sema‐3E and Plexin‐D1 mouse mutant embryos exhibit similar

vascular phenotypes, suggesting that Sema‐3E signals through Plexin‐D1 to

restrict blood vessel growth to the intersomitic boundaries. However, Plexin‐D1‐deficient mice die shortly after birth due to major defects in the cardiac

outflow tract, but Sema‐3E‐deficient mice are viable. These phenotypic diVer-ences suggest that, apart from Sema‐3E, other ligands are required for

proper cardiovascular patterning. In this term, it was proposed that morpho-

genesis of the outflow tract requires coordinated signaling of VEGF through

VEGFR‐2/Npn‐1, and of Sema‐3A and Sema‐3C through Plexin‐D1/Npn‐1and Plexin‐D1/Npn‐2, respectively (Eichmann et al., 2005).


5. Ephrins and Their EPH Receptors

Eph receptors and ephrins are membrane‐bound proteins that function as a

receptor–ligand pair, with 16 Eph receptors and 9 ephrins identified so far in

mammals. According to distinct structural properties, Eph receptors and

their ephrin ligands are classified into A and B subfamilies. Although there

is a considerable crosstalk between A and B family members, type A ephrins

preferentially bind Eph‐A receptors and type B ephrins bind Eph‐B receptors

(Heroult et al., 2006). Ephs are generally described as receptors and ephrins

as ligands, but it is now known that their interaction initiates bidirectional

signals in both the Eph‐ and the ephrin‐expressing cells (forward and reverse

signaling, respectively) (Kullander and Klein, 2002). The direct interaction

between ephrins and Eph receptors provides adhesive forces between cells,

whereas more complex interactions and coupling with intracellular signaling

molecules translate such contacts into both repulsive signals between adja-

cent cells and attractive guidance cues for cell migration (Janes et al., 2005;

Zimmer et al., 2003). It has been proposed that Eph receptors might act in a

bimodal manner, being capable of transmitting both proadhesive as well as

antiadhesive signals. In particular, reverse ephrin‐B signaling has been impli-

cated in both attractive and repulsive functions (Kullander and Klein, 2002),

suggesting that Eph‐B receptors are able to transmit both propulsive and

repulsive signals on Eph‐B/ephrin‐B interacting cells.

The Eph receptors and ephrins were first identified as repellent axon

guidance molecules in the retinotectal projection system. Axons from the

temporal retina were shown to express a high density of Eph‐A receptors and

to project to the anterior colliculus, where expression of the ephrin‐A repel-

lent is low (Carmeliet and Tessier‐Lavigne, 2005). On the other hand, axons

from the nasal retina were shown to express low Eph‐A levels and to project

to the posterior colliculus, where abundant expression levels of the ligand‐repellent ephrin‐A were detected. Later, they have been implicated in cell

migration and positioning, axonal outgrowth, axon guidance, axon fascicu-

lation, and also angiogenesis (O’Leary and Wilkinson, 1999).

Apart from the role of ephrins–Ephs in demarcating arteriovenous cell

boundaries described previously, they have also been involved in blood vessel

guidance. ECmixing experiments support amodel for the action of ephrin‐B2and Eph‐B4 in blood vessel guidance, whereby signaling via ephrin‐B2 and

Eph‐B4 leads to propulsive and repulsive eVects on ECs, respectively

(Hamada et al., 2003). In zebrafish, ephrin‐B2 is expressed in somites, where

it prevents Eph‐B3/Eph‐B4‐expressing ISVs from entering somites, thus

providing short‐range guidance cues for vessels to navigate through tissue

boundaries (Adams and Klein, 2000; Oike et al., 2002; Wang et al., 1998).

It was found that the zebrafish homologue for the C. elegans max‐1 protein,

which is mainly expressed in neuronal tissue and somites during development,

40 Zacchigna et al.

acts upstream of the ephrin pathway to regulate vascular patterning of the

ISVs (Zhong et al., 2006). Max‐1 appears to regulate membrane localiza-

tion of Ephs since, in max‐1 knockdown zebrafish cells, Eph‐B3 was shown

to form abnormal aggregates within the cytoplasm, instead of becoming

translocated to the cell surface (Zhong et al., 2006).

6. Vascular Endothelial Growth Factor

Originally identified as a growth factor able to stimulate vascular permeability

and EC proliferation, VEGF represents perhaps the best illustration of how

the crosstalk between vessels and nerves aVects development and disease.

Indeed, as described in the previous sections, VEGF is critically involved in

the development and homeostasis of various tissues and organs. This is

accomplished in part by its potent eVect on the vasculature, but certain

novel functions of VEGF, such as those influencing morphogenesis and tissue

survival, are probably independent from its ability to stimulate new vessel

growth. Over the last 5 years, a growing body of evidence has been accumu-

lated showing that VEGF has direct eVects on neural cells and is a critical

player in neurodegeneration (Oosthuyse et al., 2001; Storkebaum and

Carmeliet, 2004; Storkebaum et al., 2005). Could VEGF also have a role in

neuronal cell and axon guidance? What is known so far is that VEGF

(1) stimulates axonal outgrowth and improves the survival of cultured

superior cervical and dorsal root ganglion neurons (Sondell et al., 1999);

(2) induces neurite outgrowth from cerebrocortical neurons (Jin et al.,

2006); (3) promotes the survival of mesencephalic, hippocampal, and

cerebrocortical neurons (Jin et al., 2000; Matsuzaki et al., 2001; Silverman

et al., 1999); and (4) promotes neurogenesis in vitro and in vivo (Jin et al.,

2002). In addition, a peculiar role of VEGF in supporting the correct posi-

tioning of the facial motoneuron somata, but not of their axons, has been

established (Schwarz et al., 2004). Interestingly, Sema‐3A was shown to be

essential for guiding the axons of the same neurons, but not for their cell body

pathfinding, indicating that VEGF and Sema‐3A, instead of competing,

cooperate by patterning diVerent compartments of the same cell, to properly

guide and position the somata and the axons of facial motoneurons (Schwarz

et al., 2004). Additional evidence that VEGF can act as a chemoattractant on

neurons comes from studies on neural progenitor cells. As already mentioned

above, angiogenesis and neurogenesis occur concomitantly in the mammalian

adult DG (Palmer et al., 2000), as well as in the songbird brain (Louissaint

et al., 2002), where neurogenesis and neuronal migration are required

for structural plasticity and learning throughout adulthood (Goldman

and Nottebohm, 1983). Interestingly, clusters of proliferating cells in

these neurogenic niches were found to be positive for VEGFR‐2, whereasVEGF immunoreactivity was detected in the surrounding tissue (Palmer


et al., 2000). This raised the intriguing hypothesis that VEGF could function as

a common guidance cue to recruit both endothelial and neural progenitor cells

to the correct sites for their final diVerentiation.In retrospect, this concept should not be particularly surprising, as VEGF

and its receptors appeared first in evolution in the CNS of invertebrate

species, such as worms and flies, which lack a well‐developed vascular net-

work. In C. elegans, a family of four tyrosine kinase receptors, structurally

similar to VEGFRs, has been identified. These receptors (vascular endothe-

lial growth factor receptor or ver genes) appeared to be expressed by

specialized cells of neural origin, such as glial cells, chemosensorial neurons,

and neurons of the dorsal ganglia (Popovici et al., 2002). A platelet‐derivedgrowth factor (PDGF)/ VEGF‐like growth factor (PVF‐1) has also been

characterized in the worm, which has biochemical properties similar to the

vertebrate PDGF and VEGF, and is able to bind to the human VEGFRs

Flt‐1 and KDR, inducing angiogenesis in vertebrate model systems (Tarsitano

et al., 2006). Important questions for the future are where and when the pvf‐1gene is expressed in the worm and whether PVF‐1 binds to the four neuronal

VEGFRs. Similarly, the fruit fly also expresses a receptor tyrosine kinase

related to mammalian PDGF and VEGF receptors (PVR), which is required

for hemocytes (primitive blood cells) to migrate in response to three VEGF

hortologs (Cho et al., 2002). Loss of PVR function induces important defects

in axon tract patterning and positioning of glial cells, thus further supporting

the idea that VEGF and its receptors might have originated from the CNS

(Olofsson and Page, 2005; Sears et al., 2003). It is predictable that these small

animal model systems, devoid of a well‐established vascular network, will turn

out to be extremely powerful to unravel a possible novel eVect of VEGF, as

well as of other traditional angiogenic molecules, in axon guidance.

VII. Perspectives

Emerging evidence has highlighted the importance of the neurovascular link.

Not only blood vessels and nerves originate, develop, and branch topograph-

ically in a similar manner, but they also share common mechanisms for cell

signaling and pathfinding. Perhaps the most striking similarity is between the

growth cone of axons and the endothelial tip cells in blood vessels. Both play

a similar role in exploring the environment and function to define the

direction in which the axon or the new vascular sprout grows. Interestingly,

initial observations suggest that molecular cues, previously described as axon

guidance signals, might also be implied in angiogenesis. For instance, Netrins

or Slits seem to act as attractant or as repellent cues for ECs, but the

underlying molecular mechanisms for these eVects remain to be further

resolved: Is this dual function dependent on the cellular context or the

42 Zacchigna et al.

receptor type to which they bind? In addition, more research is required to

elucidate the intracellular pathways linking guidance receptor activation

to cytoskeletal changes in ECs. Are the signaling cascades in neuronal

guidance systems similar in ECs? Another exciting new possibility is that at

least some of the molecules involved in vessel guidance similarly regulate

axon guidance. For instance, VEGF, the key angiogenic factor, has been

recognized to be involved in many neurobiological processes, including

growth cone movement, neuronal survival, and maintenance of neuronal

circuitries, suggesting a possible role for VEGF in axon guidance.

From a therapeutic perspective, the discovery of the striking parallelism

between vessels and nerves might also pave the way for the development of

novel pro‐ and antiangiogenic therapeutic strategies. The increasing evidence

that several of the molecules involved in the pathfinding of vessels and nerves

are also expressed by diVerent tumor cells, and regulate tumor cell growth,

motility, and invasion (Klagsbrun and Eichmann, 2005), oVers new thera-

peutic concepts. Initial evidence that interfering with Robo, Sema, or ephrin

signaling inhibits tumor angiogenesis in diVerent animal models provides a

first glimpse of this therapeutic potential (Bielenberg et al., 2006; Heroult

et al., 2006; Wang et al., 2003). It also remains to be determined whether

some of these molecules will be useful for stimulating the reperfusion of

ischemic tissues in the clinic, an unmet medical need to date.

Acknowledgments

The authors thank A. Ny for kindly providing the pictures for Figs. 5A–C. Zacchigna, S. is

supported by the European Union seventh framework program via a Marie Curie intra‐European fellowship. Ruiz de Almodovar, C. is a recipient of a fellowship from the Federation

of European Biochemical Societies (FEBS).

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