Review
The Netrin family of guidance factors: emphasis on Netrin-1 signalling

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Abstract

During the development of the nervous system, neurons respond to the coordinated action of a variety of attractive and repulsive signals from the embryonic environment. Netrins form a family of extracellular proteins that regulate the migration of neurons and axonal growth cones. These proteins are bifunctional signals that are chemoattractive for some neurons and chemorepellent for others. Netrins mainly interact with the specific receptors DCC and UNC-5 family. To date, several Netrins have been described in mouse and humans: Netrin-1, -3/NTL2, -4/β and G-Netrins.

Netrin-1 is the most studied member of the family. It is involved in the development many projections of the nervous system. When Netrin-1 interacts with its specific receptors, a cascade of local cytoplasmic events is triggered. Several signal transduction pathways and effector molecules have been implicated in the response to Netrin-1: small Rho-GTPases, MAP-Kinases, second messengers and the Microtubule Associated Protein 1B (MAP1B).

Introduction

Information processing in the brain is determined, to a great extent, by an intricate network of neuronal connections. The sheer dimension of the task of connecting the neurons of the central nervous system (CNS) is amazing. In the adult human brain, each of its almost 1012 neurons connects to an average of 1000 target cells, thereby forming a specific circuit whose precise pattern is crucial for the correct function of the system. How is this pattern produced with the necessary precision and reliability during embryogenesis?

During morphogenesis of the nervous system, neurons are produced in specialized regions and then migrate through defined pathways until reaching their final location. Each neuron develops a group of dendrites that are characteristic of its phenotype and an axon that extends to form specific pathways to reach its synaptic target (Fig. 1). Precision is achieved through two kinds of processes: early neural activity-independent mechanisms and refinement mechanisms, which occur later and are activity-dependent [95], [219], [220], [235]. This review will address the role of one family of guidance molecules, the Netrin family, in activity-independent mechanisms of neuronal organization.

Neuronal migration and the guidance of axons towards their targets are regulated by mechanisms which, in many aspects, are similar to those that occur in leucocyte chemotaxis and Dictyostelium discoideum amoebae [38], [61], [66]. Neuroblasts and axons travel through the embryonic “milieu” and are guided by local signals [88] through the formation of specialized cellular structures that “explore” their surrounding environment: the growth cone in axons and the leading edge in migrating neurons [22]. These hand- or fan-like structures contain the machinery necessary to detect and respond to extracellular guidance cues and to provide the motor energy necessary for the growth of neurites. In spite of similarities, there are differences between neuroblast migration and growth cone navigation. In the former, the cell nucleus translocates in the same direction as the leading edge, retracting the posterior region of the cell body. In contrast, in growth cone navigation, the cell nucleus remains stationary and the advance of the growth cone leaves an elongated process behind it. Once the cone reaches its target, a new group of mechanisms is set in motion that allows the establishment and maturation of synaptic connections (Fig. 1) [72], [269].

Neuroblast migration and growth cone navigation occur in a stereotyped way with very few errors, even though the target is usually located quite far away. Two mechanisms guide neuroblasts and growth cones: simple linear growth along “highways” (on adhesive substrates that allow growth), alternated with more complex decision-making behaviors in intermediate targets (Fig. 2) [43], [44], [226], [235]. At very early embryonic stages, the first developing axons navigate through an axon-free environment. Later on, they grow through tissues crisscrossed by other projecting axons [9]. Developing axons and migrating neuroblasts move through fascicles of pre-existing axons, switching from one fascicle to another by a process known as selective fasciculation (Fig. 3) [195], [245]. In addition, axons and neuroblasts move towards groups of specialized cells that form the intermediary targets (or choice points; Fig. 2). These targets provide the extracellular guidance cues that regulate navigation, but they can also provide the trophic support necessary for the survival of developing axons. This mechanism allows the removal of axons that follow erroneous paths or do not reach their target [76], [249].

Guidance requires the integration of local adhesive interactions of the substrate with the directional information from guidance cues. The adhesion receptors transduce the signals from the extracellular substrate to the cytoskeleton, thereby providing the traction necessary to move the growth cone, while guidance cues supply the directional information through attraction or repulsion [36], [155], [220], [267]. However, this distinction does not appear to be so strict since adhesion molecules can also provide directional information that modifies the response to cues [28], [48], [220], [232], [267]. The growth cone integrates this information and elaborates responses, which are then translated into the reorganization of the cell cytoskeleton [220], [232], [267]. The differences in the nature of the response appear to reside more in the functional properties of the growth cone than in the identity of the signals themselves. However, it is only recently that the complex interactions that occur in the growth cone are coming to light.

Growing neurites respond to the coordinated action of a great variety of attractive and repulsive signals [13], [14], [23], [24], [95], [222], [235], [236]. The term “attractive” refers to effects that range from allowing growth to attraction. The response to negative cues extends from a simple deviation of the growing axon to the collapse and retraction of the growth cone. In addition, these signals can be soluble, thereby acting over large distances, or be bound to cell membranes or to the extracellular matrix, operating by contact over short distances (revised in [235]; Fig. 4). However, many molecules do not adjust to a single guidance mechanism. Instead, it is the growth cone that interprets the signal as attractive or repulsive. Furthermore, a diffusible molecule may act over a short distance, restricting its diffusion by interaction with the extracellular matrix, or a molecule anchored to substrate can be expressed through gradients, thereby acting as a long-distance chemotrophic signal [41], [43], [49], [55], [63], [95], [123], [169], [209], [212], [220], [234], [235], [267].

Many molecules have been implicated in guidance, including growth-promoting factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECMs). Some of these include Reelin, Neurotrophins, growth factors such as HGF (Hepatocyte Growth Factor/Scatter Factor) and members of the TGF-β family (Transforming Growth Factor β). Other candidates include the family of protocadherins, odorant receptors, Ig-CAMs and neurexins [7], [8], [36], [59], [67], [74], [162], [173], [248], [267]. In addition to these molecules, genetic, biochemical and molecular approaches have identified four conserved families of guidance signals: Netrins, Slits, Semaphorins and Ephrins. One or more receptors have been identified for each of these cues: Deleted in Colorectal Cancer, DCC (Unc40) and UNC-5, Robo, Neuropilins and Plexins, and Ephs, respectively [18], [38], [50], [55], [78], [134], [151], [173], [176], [180], [190], [194], [229], [255], [267].

Finally, growing axons and neuroblasts must select and enter their final targets [88], [110], [245]. Once there, axons grow to determined topographic locations or cell layer. Very often, the target establishes gradients and distinct layer-distribution of local guidance cues that control the position of the developing axons [79], [87], [110], [175], [186], [261], [262]. When the growth cone finds its correct synaptic target, the terminal region of the axon branches and establishes synapses. The selection of post-synaptic cells, or of precise membrane domains within these cells, may involve several molecules that could be organized in a combinatorial code [69], [110], [119], [172], [235], [243]. Several studies have proposed that the selection of the correct target area is independent of the formation of synapses [110], [191], [192], [262]. Finally, neuronal activity drives the refining of axonal projections, although this takes place after synapse formation.

Section snippets

The Netrin family

For decades, developmental neurobiologists searched for the chemoattractive molecules that Ramón y Cajal described as fundamental axonal guidance mechanisms [22]. The first molecules were identified in chick embryo through biochemical purification of growth-promoting factors for commissural axons in the spinal cord [124], [208]. These were named Netrins (“one who guides” in Sanskrit). Two proteins (Netrin-1 and -2), of about 80 kD and with 78% of homology, were isolated. These proteins are

Netrin receptors

The attraction and repulsion responses produced by Netrin-1 are mediated by two receptor families: DCC and UNC-5 proteins [99], [151]. These two families belong to the immunoglobulin superfamily and cross the membrane only once [56], [111]. The DCC receptors mediate mainly attraction but also participate in repulsion (Fig. 6, Fig. 11). UNC-5 receptors appear to act in repulsion only, alone or in combination with DCC receptors. UNC-5 receptors may require the participation of DCC as a

Signalling pathways activated by Netrin-1

One hundred years after that Ramon y Cajal described the importance of the growth cone [22], numerous experiments have shown that its motility and guidance depend on the subjacent actin and microtubule cytoskeleton [63], [131], [220], [228]. When a guidance signal interacts with its specific receptor on the surface of the growth cone, a cascade of cytoplasmic events is triggered (Fig. 12), thereby promoting the reorganization of the associated cytoskeleton to generate a range of responses to

Receptors as the combinatorial sum of modules

How can the binding receptor-ligand produce such distinct functions? The response of a receptor after integration with its ligand is determined by the: (a) interaction of distinct receptors to form a multimeric receptor complex; (b) domains of each receptor that are activated within this complex and (c) intracellular molecules that couple to the activated receptor, thereby triggering distinct intracellular transduction pathways [267].

Receptors should be considered units comprised of modules or

Acknowledgments

We thank T. Yates for editorial assistance. This study was supported by grants from MCYT (SAF01-3098, SAF01-134, SAF2004-07929), The Caixa Foundation and The Marató de TV3 Foundation to E.S., and from FIS (01-0895) to J.A.D.R.

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