Molecular Mechanisms Contributing To Axon Guidance Biology Essay

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Abstract

The optic pathway at present represents the best model to understand the molecular mechanisms contributing to axon guidance. In recent years a great number of interactions have been shown to play a role in this process.

This review will give a broad overview of the steps involved in the development of the optic pathway, focusing in particular on the Slit-Robo signalling system.

Introduction

The visual perception of the surrounding environment is transmitted from the eye to the central nervous system (CNS) through the retinal ganglion cells (RGCs).

The correct advancement of RGCs axons during development from the retina to the central nervous system (CNS) involves the binding of receptors located in their plasma membranes with a variety of guidance cues. These ligand-receptor interactions as well as the subsequent intracellular signalling are modified by the action of regulatory genes and post-transcriptional mechanisms.

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The optic pathway is at present the most helpful model in the research on axon guidance, due to its anatomical position, easy manipulation and the constant recurrence of its projection 1.

The first part of this review will give a general overview on the development of the optic pathway while the second part will focus on the role of Slit, a specific ligand involved in this process, and its receptor Robo.

The development of the optic pathway

During development RGCs differentiate within the retina before lengthen their axons radially towards the optic nerve head (figure 1) 1. In doing so, RGCs form the inner cellular layer of the retinal tissue, known as optic fibre layer. After having entered the optic nerve, RGCs axons extend to build the optic chiasm. At this point, in animals possessing stereovision, the temporal axons project ipsilaterally while the ones generating from the nasal retina cross the midline towards the contralateral part of the diencephalon or the midbrain. Both crossing and non-crossing axons continue their journey within the tractus opticus before reaching either the lateral geniculate nucleus (LGN) contained in the developing thalamus or other targets within the central nervous system such as the superior colliculus (SC).

Figure 1: schematic representation of the visual pathway in mammals 1. LGC: lateral geniculate nucleus, SC: superior colliculus, D: dorsal, L: lateral, M: medial, N: nasal, T: temporal, V: ventral.

The extension of the retinal ganglion cells' axons from the retina to their final target within the CNS is regulated by a multitude of guidance clue. These molecules usually either attract or repel the axon. Some cues, however, have been shown to have the ability to generate both reactions, depending on the developmental stage and position of the neural fibres 2.

The distal extremity of the axon, known as growth cone, contains specific receptors which interact with the guidance cues which act as ligands. Following complex intracellular cascades the axon adjusts its direction accordingly. The first phases of the axon's movements are produced by the extension or retraction of the filopodia and lamellipodia present on the top of the growth cone. The progress of these highly dynamic structures containing F-actin bundles is followed by the advancement of the entire axon. The main structural elements of the axonal shaft, on the other hand, are the microtubules (figure 2) 1.

Figure 2 2: A: structure of the growth cones (see text). B, C: the advance of the axon follows the movements of the filipodia and lamellipodia after interaction with repellent and attractant clues 1.

Different ligand (clues)-receptor mechanisms which are involved at different stages of the axon's journey have been described in recent years. Under others, these include Ephrins/Ephs 3, Slits/Robos 4, netrin/Dcc/UNC5 5 and Semaphrins/Neuropilins/Plexins 6.

Regarding the interaction between Ephs and Ephrins, the dissimilar concentration's gradients of the latter along the optic pathway produces the precise mapping of the axons in the LGN and SC 7, 8.

Slits, on the other hand, adjust the direction of the axons in the retina, preventing their growth into other layers than the outer fibre layer (OFL) 9. In addition, these clues also inhibit the misdirection of axons at the level of the chiasma and of the optic tract 10.

Moreover, netrin-1 is expressed in the optic disc where it regulates the penetration of the axons from this structure into the optic nerve 11.

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Finally, Sema3D is produced in the midline of the ventral diencephalon, inducing the projection of axons to the contralateral side at the level of the chiasm 12.

In addition to the receptor-ligand signalling system, also secreted factors such as Sonic hedgehog, FGFs, Wnts and BMPs play an important role in the axon guidance 1.

Wnt3, for example, is expressed in the tectum, contributing to the mapping in this region of the CNS 13.

These are only some examples of the clues' role in the formation of the optic pathway.

The retinal development as well as the axonal progression is regulated by the expression of specific regulatory genes and transcription factors which often interact with receptors and guidance cues 1. These include Pax2, Vax1, Vax2 and FoxG1 which in mutant mice have been shown to indirectly lead to aberrant axonal trajectories 1, 14, 15.

Other mediators such as Zic2, Zic3, Brn3b have also been reported to cause a direct modification in the axonal direction 1, 16. Zic2, in particular, induces the RGCs in which it is expressed at the level of the chiasma to take an ipsilaterally direction 17.

Finally, a mutation of SOHo, GH6, FoxD1 or FoxG1 in the chick retina alters the expression of EphAs, resulting in aberrant axonal projections into the tectum 1, 18.

The expression of both receptors and guidance clues is also regulated by different types of post-transcriptional regulators. These include mechanisms which lead to alterations in the receptor-ligand interactions (i.e. heparan sulphate proteoglycans and stromal cell-derived factor-1), modifications in the cytoplasmatic concentration of second messengers (i.e. cyclic nucleotides) and changes in the concentration of receptors, for example through endocytosis 1.

The Slit-Robo signalling system

Most of the times, the interaction between Slit (ligand) and Robo (receptor) causes a repulsion of the growth cones 9, 10. While in the retina this signalling system prevents the growth of the RGCs axons outside the OFL, within the CNS, it avoids the aberrant crossing of the nervous fibres across the midline 9, 10.

After having explained the structural elements which build this signalling system, I will briefly elucidate the role of the Slit-Robo complex in the optic pathway formation.

While invertebrates possess only one Slit, vertebrates express three different forms of this molecule (Slit1-Slit3). At the N-terminus, Slit consists of four leucine-rich repeat regions (LRR), known as D1-D4. These are followed by a series of domains as clearly pictured in figure 3 (Drosophila): six epidermal growth factors (EGF), one laminin G-like and a C-terminal cysteine knot domain 19.

On the other hand, three types of Robos exist in Drosophila and four in vertebrates 19. The extracellular Robo is made of five immunoglobulin-like (IG) regions and three fibronectin type 3 (FN3) domains. The intracellular segment of this receptor consists of four well conserved domains known as CC0-CC3 and a large series of variable regions (figure 3) 19.

In both Drosophila and vertebrates, Slits D2 interacts with Robo IG1 in order to produce a signalling cascade (figure 3 and 4) 20, 21. As shown in previous studies, deleting IG1 or IG2 prevents the interaction with Slit2 while IG3-IG5 and FN3 are not needed in the binding process 19.

Figure 3 19: schematic representation of the Slit-Robo complex in Drosophila.

Figure 4 19: reconstruction of the Slit-Robo complex.

Heparan sulphate (HS) is essential in the Slit-Robo interaction 19. This ligand/receptor signalling was shown to be blocked in vitro as well as in vivo in models lacking HS 22, 23.

It is still not completely clear how the Slit-Robo complex leads to the intracellular cascade which results in a structural change of the cytoskeleton and in the subsequent alteration of the axon's trajectory. It is currently believed that adaptors bound at the intracellular segment of Robo are modified after the Slit-Robo interaction following not yet completely understood mechanisms. This would then result in a cytoplasmatic signalling cascade 19. For example, following the binding of Slit to Robo, srGAP1 (Slit-Robo Rho GTPase-activating protein 1) has been reported to combine with CC3, causing the blockage of Cdc42 (cell division cycle 42) activity. The latter is a GTPase belonging to the Rho family 24 and its alteration could induce one of the possible cascade pathways 19.

The Slit-Robo interaction is essential for an appropriate development of the optic pathway.

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First of all, this signalling mechanism prevents the progression of RGC axons into retinal layers other than the optic fibre layer (OFL). In vitro, Slit1 and Slit2 have been shown to block the axon's outgrowth 25. Similarly, in mice not expressing Slit some RGC axons penetrate into deeper retinal layers 9. Slit1, however, in chick retina has also been shown to attract RGC axons 26. This means that Slits can fulfil attractive as well as inhibitory functions 1. On the other hand, after being released by the lens, Slit2 has only been reported to direct the axons towards the optic disc through inhibition of the growth cone 1.

Slit cues are also released at the level of the optic chiasm. Similarly to the retina, their main role is to guide the RCG axons in the correct formation of the chiasm 25. More specifically, in Drosophila Slits inhibit the midline's crossing of those axons which are meant to project ipsilaterally while preventing contralaterally directed fibers from crossing again 27. Mutations in both Drosophila Robo and Slits lead to aberrant midline crossing of the RGC axons 27.

Finally, Slits molecules and Robo receptors are expressed along the optic tract where they prevent the progression of the neural fibres into regions other than their target structures 13.

Conclusion

The research studying the development of the optic pathway has given a fundamental contribution to the understanding of the molecular mechanisms controlling axonal guidance. In addition to the ocular system, these biological steps may help us also to clarify the growth of axons in other organs such as the central and the peripheral nervous system.

Further studies, however, are needed to reveal the complexity of the interactions and cascades occurring during this formation's process.