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Rôle des interactions mécaniques entre tissus dans la mise en place du circuit olfactif du poisson-zèbre
Role of chemical cues in neuronal circuit formation
Diversity of neuronal guidance mechanisms
After neuronal progenitors have migrated to their final location in the organism, they start to emit multiple and undifferentiated protrusions. One of them will specify into the axon and extend in the direction of its target, to which it will connect and form a synapse. Chemical cues driving this process of axon elongation and guidance have been studied since the late th century when Cajal first discovered “a cone-like lump with a peripheral base” at the tip of the extending axon. He called this specialized, dynamic, and cytoskeletal-rich structure the growth cone. As an attempt to explain how a nerve cell develops, forms its connections and displaces its cell body, he hypothesised that the growth cone might be guided by chemical cues in the extracellular environment (Cajal, 09; Garcia-Marin et al., 09). Over a 100 years of research confirmed the hypothesis that growth cones are able to select the correct path towards their target by responding to the appropriate set of cues, and revealed a variety of axon guidance mechanisms (as illustrated in Figure 2). Chemical cues can operate either at short or long range: short range guidance requires direct contact between the growing axon and the cells that provide the guidance cue (Brankatschk and Dickson, 06) while the distance between the source of long range cues and their target may vary between a few cells to a whole tissue (Wu et al., ). Chemical guidance cues can be either chemoattractive or chemorepulsive. They are capable of regulating interactions between axons and their underlying substrates but also of influencing the bundling or unbundling of axons together into nerves, through fasciculation or defasciculation processes (Kolodkin and Tessier-Lavigne, 11). Figure 2 : Schematic illustration of the diversity of neuronal guidance mechanisms. Neuronal processes are guided by chemical cues that can function at long and short distances to mediate either attractive or repulsive guidance. (Kolodkin and Tessier-Lavigne, 11) This variety of mechanisms is associated with a range of axon guidance molecules among which Netrins, Slits, Semaphorins, and Ephrins represent conserved well-known families of proteins (Dickson, 02; Kolodkin and Tessier-Lavigne, 11).
Conserved families of guidance molecules
Netrins are a small family of phylogenetically conserved secreted proteins. They have initially been identified as chemo-attractants for commissural axons in chick embryos and rat explants (Kennedy et al., 94) and for circumferential axon guidance in Caenorhabditis elegans (Hedgecock et al., 90). Recent studies showed that Netrins can function as both short-range and long-range guidance cues, even within the same system (Wu et al., ). Netrin receptors DCC and Unc5 participate in both attraction and repulsion, or only in repulsion, respectively. Slits are large secreted proteins that signal through Roundabout (Robo) family receptors. Slits have been first implicated in axon guidance when searching for an axon repulsive cue in Drosophila midline (Kidd et al., 99). Slits, like Netrins, are bifunctional, since they are also involved in promoting axon elongation in other contexts, as shown in vitro (Wang et al., 99). Semaphorins are a large, phylogenetically conserved family of proteins divided into eight classes, on the basis of their structure: classes 1 and 2 are found in invertebrates, classes 3 to 7 in vertebrates, and class V semaphorins in viruses (V for virus, not represented in Figure 3A). As shown in Figure 3A, some semaphorins are secreted guidance molecules while others are transmembrane proteins. Semaphorin role in axon guidance has been first shown in grasshopper (Kolodkin et al., 92). Many Semaphorins can function as both attractive and repulsive cues, and even the same Semaphorin may under certain circumstances serve in both capacities (Dickson, 02). Semaphorins signal through multimeric receptor complexes during the establishment and maintenance of neuronal connectivity. The variability of potential receptors suggests that the function of Semaphorins, either as repellents or attractants, could actually depend on the use of different receptor complexes and thus the activation of distinct intracellular signalling pathways within the growing axon (Kolodkin and Tessier-Lavigne, 11). Ephrins are cell-surface signalling molecules divided into two classes: Ephrin-As, which are anchored to the membrane by a glycosylphosphatidylinositol linkage and bind EphA receptors; and Ephrin-Bs, which have a transmembrane domain and bind EphB receptors. Their role in axon growth and axon pathfinding has been first discovered in cultures of vertebrate retinal ganglion cells (RGCs) where Ephrin induced growth cone collapse and repulsion of RGC axons (Drescher et al., 95). Like the other three families of chemical cues, Ephrins can mediate either attraction or repulsion. Because Ephrins do not appear to be active when not bound at the cell surface, they probably function only as short range guidance cues, which is not the case for the three other families that have been found to function both as short-range signals, staying in close proximity with the cells that produced them, and as long-range signals, diffusing from their source (Kolodkin and Tessier-Lavigne, 11). Figure 3: Conserved families of guidance molecules (A) and their receptors (B). (Dickson, 02) The four families described above illustrate the diversity of chemical cues driving axon growth, pathfinding and fasciculation. When the axon meets these guidance molecules, different signalling pathways are activated, most of them involving the small RhoGTPase proteins, RhoA, Rac and Cdc42, which in turn control the cytoskeleton organization (Lowery and Van Vactor, 09). The recognition of these molecules, the activation of the downstream signalling cascades and finally the direction change mainly occurs in the axon tip, the growth cone (Mortimer et al., 08; Short et al., ).
The growth cone
Not only does the growth cone translate environmental signals into directional movement – a function called “navigator” in (Lowery and Van Vactor, 09) – but it also acts as a motor for the rest of the axon as it propels the axon shaft forward – a function called “vehicle”. To give a picture, the growth cone can be seen as both the steering wheel and the engine of the extending axon. In order to achieve these functions, the growth cone is composed of dynamic cytoskeletal components that determine its shape and movement on its journey. At its distal edges are fingerlike filopodia, narrow cylindrical membrane extensions capable of extending tens of microns from the periphery of the growth cone. They are separated by sheets of membrane called lamellipodia-like veils (Lowery and Van Vactor, 09) (Figure 4a). The growth cone can be separated into three domains based on the cytoskeletal distribution: the peripheral (P) domain, composed primarily of lamellipodia and filopodia; the central (C) domain, invested by stable, bundled microtubules that enter the growth cone from the axon shaft, in addition to numerous organelles and vesicles, and the transitional (T) domain at the interface of the P domain and the C domain (Dent and Gertler, 03) (called periphery, transition zone and central zone on Figure 4a). In response to attractive or repulsive chemical cues, axons exert mechanical forces on their environment in order to move: this is the vehicle function of the growth cone. K. Franze reviewed the intrinsic forces driving growth cone motility and suggested the following model (Figure 4). The addition of new actin monomers to the growing barbed ends of actin filaments in the filopodia results in a compressive polymerization force, FP in the P domain (Figure 4b,c). At the same time, in the C domain, myosin II motors pull on the actin filaments, generating a contractile force FC. The myosin-mediated contractile forces, FC, and the actin-mediated polymerization forces, FP lead to a constant retrograde flow of actin filaments away from the P domain (where actin polymerizes) towards the C domain (where actin depolymerizes). Moreover, actin filaments in the growth cone are coupled to the extracellular environment by so-called point contacts, composed of a large number of adaptor and signalling proteins such as cell-adhesion molecules (CAMs) serving as molecular clutches. It is via these clutches that the forces generated within the growth cone are transmitted to the surrounding environment. At low clutch engagement, there is a fast retrograde actin flow and the growth cone does not move much. At high clutch engagement, retrograde actin flow slows down, actin polymerization pushes the membrane forward, the traction force of the growth cone on its substrate, FT, increases, and the growth cone advances. Thus, the growth cone advance depends on the level of clutch engagement, enabling fast adaptation to changes in the environment (Franze, ).
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