Growth cones: Tropic cues involved in growth cone guidance

6:03 AM | BY ZeroDivide EDIT

Fig.1: Various tropic cues interact to guide the growth cone; the initial role of these cues was identified as either repulsive (red) or attractive (green).
The growth cone is a highly dynamic structure of the developing neuron, changing directionality in response to different secreted and contact-dependent guidance cues; it navigates through the developing nervous system in search of its target. The migration of the growth cone is mediated through the interaction of numerous trophic and tropic factors; Netrins, Slits, Ephrins and Semaphorins are four well-studied tropic cues (Fig.1). The growth cone is capable of modifying its sensitivity to these guidance molecules as it migrates to its target; this sensitivity regulation is an important theme seen throughout development.

Netrins[edit]

Fig.2: Netrin signaling has multiple roles in guidance.
Netrins are diffusible chemoattractive molecules that guide commissural axons across the midline; they are secreted by floor plate cells at ventral midline of the spinal cord.[1]Netrins establish a gradient to direct commissural axons at a distance; Netrin-2 is expressed broadly in the ventral two thirds of the spinal cord, but not in the floor plate. Mice with Netrin-1 loss-of-function exhibit severe disruption in commissural axon migration; this experiment established the importance of Netrin-1 in guidance decisions.[2]
Netrin-1 gradient in Xenopus laevis ganglion cell can induce turning of retinal growth cones in vitro to steer axons out of the retina.[3] Netrin (unc-6, Caenorhabditis eleganshomologue) and its corresponding receptor DCC (Deleted in Colorectal Cancer) were initially identified as an attractive interaction.[4] DCC, expressed by commissural axons, binds to Netrin with high affinity; inhibiting Netrin/DCC signaling interferes with the attractive turning of retinal growth cones.[3]
Netrin-1 has also been shown to act as a chemorepellent in vivo for trochlear motor axons that migrate dorsally away from the floor plate.[5] Interestingly, in Netrin-1 deficient mice, trochlear axon projections are normal,[2]suggesting the existence of other redundant guidance cues working in tandem with Netrin-1 to repel trochlear axons.
Studies in C. elegans revealed a possible mechanism for Netrin acting as a chemorepulsive agent (Fig.2). Unc-5, a transmembrane protein, is required for dorsal migration of axons in nematodes; it was determined that unc-5 acts as a repulsive receptor for Netrin (unc-6). The switch between attractive and repulsive Netrin signaling can be mediated by misexpression of unc-5 in commissural axons.[6] Netrin-1/DCC binding induces DCC homodimerization leading to an attractive response; on the other hand, the chemorepellent response is triggered via Netrin-1 binding to unc-5/DCC heterodimers.[7]
Netrin repulsion can also be mediated by changes in cyclic nucleotide levels; Netrin-1 induces a repulsive response when cAMP signaling is inhibited.[8] Cis interactions of Netrin/DCC (attractive) and Slit/Robo (repulsive) in commissural axons silence both signaling cues; this illustrates how multiple tropic cues interact to guide the commissural axons to their targets.[9]

Slits[edit]

Fig.3: Drosophila and vertebrates use different mechanisms to regulate their sensitivity to Slit mediated repulsion at the midline. Drosophila regulate their sensitivity through Comm mediated endosomal trafficking of Robo (top panel); vertebrates use alternatively spliced Robo3 isoforms to regulate Slit signaling (bottom panel).
Repulsive cues play an important role in guiding growth cones to their appropriate target; roundabout (Robo) receptors and their ligand, Slit, are a well-studied example of repulsive guidance. Robo receptors were initially identified in Drosophila melanogasterusing a forward genetic screen to search for molecules involved in midline crossing at the floor plate.[10][11] Robo/Slit loss-of-function mutations result in axons crossing the midline multiple times, whereas gain-of-function results in little to no midline crossing; consequently this interaction was determined to be important in preventing non-commissural axons from crossing the midline and commissural axons from recrossing.
How the commissural axon regulates its response to Slit repulsion at the midline has been extensively studied in both Drosophila and vertebrates; in these two models the growth cone’s response to Slit has been shown to be regulated through receptor trafficking and alternative splicing, respectively (Fig.3).[12][13]
Receptor trafficking is used extensively throughout growth cone migration; inDrosophila prior to crossing the midline these neurons express commissureless (comm), a protein involved in Robo receptor trafficking. Comm prevents Robo from reaching the cell membrane by targeting the receptor for the endosomal pathway; this allows the growth cone of the commissural axon to cross the midline by preventing Robo/Slit repulsive interactions. Comm expression turns off after the growth cone has crossed the midline; this permits Robo/Slit repulsion and prevents the growth cone from crossing the midline again.
Vertebrates, on the other hand, do not possess a comm homolog; instead they facilitate midline crossing through alternative splicing of Robo3 (aka.Rig-1). Robo3 has two isoforms, 3.1 and 3.2, and these isoforms interact with Robo1 and Robo2 (Robo1/2) through cis interactions at the leading edge of the growth cone. Before crossing the midline Robo3.1 inhibits Slit/Robo repulsive signaling, allowing the commissural axon to cross; after crossing the midline Robo 3.1 is replaced by Robo3.2 to facilitate the repulsive Slit/Robo signaling through cis interactions with Robo1/2.
Slit/Robo signaling is seen throughout the developing nervous system and is demonstrative of the importance of repulsive cues in growth cone migration; the aforementioned regulation of these repulsive barriers determines the path of the commissural axon.

Ephrins[edit]

Fig.4: Ephrin/Eph bidirectional signaling; forward signaling (ligand to receptor) and reverse signaling (receptor to ligand).
In the 1940s Roger Sperry was conducting experiments on newts and frogs to understand how axons are guided to their topographic locations; he did this by cutting the optic nerve and rotating the detached eye 180°. What he observed was that the animals behaved as if their visual world was back-to-front and upside-down when presented with a lure in front of them.[14] He explained this behavior by the existence of two or more gradients "that spread across and through each other with their axes roughly perpendicular"; this became known as the chemoaffinity hypothesis. This subsequently lead to extensive research and the discovery of two repulsive factors, Ephrin-A5 and Ephrin-A2, by observing axon growth in retinal tissue culture on a striped carpet of anterior and posterior tectum membrane.[15][16]
Ephrins are divided into 2 classes: Ephrin-As are bound to the membrane through GPI (glycosylphosphatidylinositol) linkage and Ephrin-Bs have a transmembrane domain and a short cytoplasmic domain; they interact with their respective receptors Eph-A and Eph-B which are members of the tyrosine kinase family. One unusual feature about Ephrins is their ability to bidirectionally signal (Fig.4); they can participate in both forward (ligand to receptor) and reverse signaling (receptor to ligand). Eph/Ephrin binding induces conformational changes in the Ephrin transmembrane and cytoplasmic domains, activating the signaling pathway. Eph/Ephrin forward signaling regulates actin dynamics via small GTPases of the Rho family; reverse signaling occurs when the Ephrin-B cytoplasmic tail gets phosphorylated at tyrosine residues. Ephrin-B also contains a PDZ binding motifimportant in axon guidance regulation via G-protein signaling. Reverse signaling can also occur when Ephrin-A is activated by Eph-A3 binding; this is regulated by metalloprotease-dependent cleavage of Ephrin-A.[17]
Eph/Ephrin bidirectional signaling is important for axon guidance and target selection; mapping of retinal axons along anterior-posterior axis in the visual system is regulated by Ephrin-A/Eph-A mediated repulsion. In the tectum, the transcription factor Engrailed creates an Ephrin-A concentration gradient along the anterior-posterior axis; this results in different signaling cues to the growth cones which also express graded levels of the receptor. Interestingly, the topographic location of retinal axons along dorsal-ventral axis requires both forward and reverse signaling by Ephrin-B/Eph-B gradient mediated attraction.[9]
As described above for the visual system, Eph/Ephrin signaling plays an important role in topographic mapping in several other regions of the developing nervous system; the bidirectional signaling illustrates some of the complex regulatory mechanisms involved in growth cone guidance and target selection.

Semaphorins[edit]

Fig.5: Semaphorins can exist either in a soluble form or membrane bound form. Both are utilized in axonal repulsion, but recent studies demonstrate that Semaphorins can also mediate attraction. In addition, soluble Sema3A binds to Neuropilin-1, which lacks an intracellular domain and thus co-signals with Plexin A. Sema4A in its membrane form binds to Plexin B1 for its signaling pathway. Together, these mammalian Semaphorins can mediate axonal guidance and target selection.
Semaphorins are a family of chemical signaling molecules involved in axonal targeting and guidance. Sema3 was the first vertebral Semaphorin discovered,[18] and since then Semaphorins have been shown to elicit both attraction and repulsive responses in commissural axons; additionally, Semaphorins can function as a secreted or contact-dependent guidance cue (Fig.5).
Semaphorins have also been shown to mediate other neuronal processes besides targeting such as: apoptosis,[19] cell migration,[20] axon pruning,[21] synaptic transmission,[22] and axonal transport.[23] Semaphorins are the main ligands for theNeuropilin 1 (Npn1) receptor; this receptor is typically located in the medial and lateral portions of the lateral motor column during the early embryonic period of motor neuron development.[24] Upon binding Semaphorins, the Npn1 receptor transmits signaling to adjacent surface molecules, known as Plexins; this is necessary because the Npn1 receptor lacks an intracellular domain.[25] The intracellular signaling mediated through Semaphorins results in growth cone collapse, guidance, and turning;[26] this intracellular signaling is transduced through Rho family GTPases, which act to remodel the cytoskeleton of the cell.[27] In addition, several other cell surface molecules have been shown to interact with secreted Semaphorins. One example is the Ig cell-adhesion molecule (IgCAM) family; this family of adhesion molecules are suggested to interact with Semaphorins to fine tune their axonal projections and targeting.[28] The multitude of molecules that complex with Semaphorins may be the result of the ubiquitous nature of Semaphorin expression in vertebrates.
During embryonic neurodevelopment, synapse elimination and axonal pruning are critical to ensure normal functioning of the central and peripheral nervous system. Studies have suggested that Sema3A/Neuropilin 2 (Npn2) interactions mediate synapse elimination and axonal pruning, as demonstrated by Sema3A/Npn2 loss-of-function studies in mice.[21] The attractive cues mediated by Semaphorins are not well understood at the moment; however, protein kinase focal adhesion kinase (FAK) and MAP Kinase (MAPK) have been implicated in mediating downstream attractive signaling upon Semaphorin receptor stimulation.[29]
Semaphorins also play a critical role in cranial nerve development; studies using mice deficient in Sema3A and Sema3F have resulted in abnormal cranial nerve extension and defasciculation,[30] while Sema3F has been suggested to be required in order to establish projections of cranial nerves.[31] Mice deficient in membrane bound Sema6A showed misprojection of corticothalamic fibers and axon projections from the hippocampus to olfactory bulb.[31] Knocking out certain members of Semaphorins, such as Sema5A, resulted in embryonic lethality in mice and thus it has been difficult to elucidate the role of Sema5A in neurodevelopment.[32] While the previous example suggested that Semaphorins may play pivotal roles in maintaining viable neurons, for the most part Semaphorin knockout animals display mild phenotypes.[33]
Scientists thus hypothesize that there is considerable redundancy of Semaphorin family types.[33] Future studies may focus on the implication of Semaphorins in neurologic diseases, and thus developing synthetic versions of Semaphorin receptor agonists/antagonists could be beneficial for both embryonic and adult neurologic dysfunction.

References[edit]

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From Wikipedia, the free encyclopedia

Image of a fluorescently-labeled growth cone extending from an axon F-actin (red) microtubules(green).
A growth cone is a dynamic, actin-supported extension of a developing axon seeking its synaptic target. Their existence was originally proposed by Spanish histologist Santiago Ramón y Cajal based upon stationary images he observed under the microscope. He first described the growth cone based on fixed cells as “a concentration of protoplasm of conical form, endowed with amoeboid movements” (Cajal, 1890).[1] Neuronal growth cones are situated on the very tips of nerve cells on structures called axons and dendrites. The sensory, motor, integrative, and adaptive functions of growing axons and dendrites are all contained within this specialized structure.

Structure[edit]

Two fluorescently-labeled growth cones. The growth cone (green) on the left is an example of a “filopodial” growth cone, while the one on the right is a “lamellipodial” growth cone. Typically, growth cones have both structures, but with varying sizes and numbers of each.
The morphology of the growth cone can be easily described by using the hand as an analogy. The fine extensions of the growth cone are known as "filopodia" or microspikes. The filopodia are like the "fingers" of the growth cone; they contain bundles of actin filaments (F-actin) that give them shape and support. Filopodia are the dominant structures in growth cones, and they appear as narrow cylindrical extensions which can extend several micrometres beyond the edge of the growth cone. The filopodia are bound by membrane which contains receptorsand cell adhesion molecules that are important for axon growth and guidance.
In between filopodia--much like the webbing of the hands--are the "lamellipodia". These are flat regions of dense actin meshwork instead of bundled F-actin as in filopodia. They often appear adjacent to the leading edge of the growth cone and are positioned between two filopodia, giving them a “veil-like” appearance. In growth cones, new filopodia usually emerge from these inter-filopodial veils.
The growth cone is described in terms of three regions: the peripheral (P) domain, the transitional (T) domain, and the central (C) domain. The peripheral domain is the thin region surrounding the outer edge of the growth cone. It is composed primarily of an actin-based cytoskeleton, and contains the lamellipodia and filopodia which are highly dynamic. Microtubules, however, are known to transiently enter the peripheral region via a process called dynamic instability. The central domain is located in the center of the growth cone nearest to the axon. This region is composed primarily of a microtubule-based cytoskeleton, is generally thicker, and contains many organelles and vesicles of various sizes. The transitional domain is the region located in the thin band between the central and peripheral domains.
There are also many cytoskeletal-associated proteins, which perform a variety of duties within the growth cone, such as anchoring actin and microtubules to each other, to the membrane, and to other cytoskeletal components. Some of these components include molecular motors that generate force within the growth cone and membrane-bound vesicles which are transported in and out of the growth cone via microtubules. Some examples of cytoskeletal-associated proteins are Fascin and Filamin (actin bundling), Talin (actin anchoring), myosin (vesicle transport), and mDia (microtubule-actin linking).

Axon branching and outgrowth[edit]

The highly dynamic nature of growth cones allows them to respond to the surrounding environment by rapidly changing direction and branching in response to various stimuli. There are three stages of axon outgrowth, which are termed: protrusion, engorgement, and consolidation. During protrusion, there is a rapid extension of filopodia and lamellar extensions along the leading edge of the growth cone. Engorgement follows when the filopodia move to the lateral edges of the growth cone, and microtubules invade further into the growth cone, bringing vesicles and organelles such as mitochondria and endoplasmic reticulum. Finally, consolidation occurs when the F-actin at the neck of the growth cone depolymerizes and the filopodia retract. The membrane then shrinks to form a cylindrical axon shaft around the bundle of microtubules. Axon branching also occurs via the same process, except that the growth cone “splits” during the engorgement phase.
Overall, axon elongation is the product of a process known as tip growth. In this process, new material is added at the growth cone while the remainder of the axonal cytoskeleton remains stationary. This occurs via two processes: cytoskeletal-based dynamics and mechanical tension. With cytoskeletal dynamics, microtubules polymerize into the growth cone and deliver vital components. Mechanical tension occurs when the membrane is stretched due to force generation by molecular motors in the growth cone and strong adhesions to the substrate along the axon. In general, rapidly growing growth cones are small and have a large degree of stretching, while slow moving or paused growth cones are very large and have a low degree of stretching.
The growth cones are continually being built up through construction of the actin microfilaments and extension of the plasma membrane viavesicle fusion. The actin filaments depolymerize and disassemble on the proximal end to allow free monomers to migrate to the leading edge (distal end) of the actin filament where it can polymerize and thus reattach. Actin filaments are also constantly being transported away from the leading edge by a myosin-motor driven process known as retrograde F-actin flow. The actin filaments are polymerized in the peripheral region and then transported backward to the transitional region, where the filaments are depolymerized; thus freeing the monomers to repeat the cycle. This is different from actin treadmilling since the entire protein moves. If the protein were to simply treadmill, the monomers would depolymerize from one end and polymerize onto the other while the protein itself does not move.
The growth capacity of the axons lies in the microtubules which are located just beyond the actin filaments. Microtubules can rapidly polymerize into and thus “probe” the actin-rich peripheral region of the growth cone. When this happens, the polymerizing ends of microtubules come into contact with F-actin adhesion sites, where microtubule tip-associated proteins act as "ligands". Laminins of the basal membraneinteract with the integrins of the growth cone to promote the forward movement of the growth cone. Additionally, axon outgrowth is also supported by the stabilization of the proximal ends of microtubules, which provide the structural support for the axon.

Axon guidance[edit]

Main article: Axon guidance
Model of growth cone-mediated axon guidance. From left to right, this model describes how the cytoskeleton responds and reorganizes to grow towards a positive stimulus (+) detected by receptors in the growth cone or away from a negative stimulus (-).
Movement of the axons is controlled by an integration of its sensory and motor function (described above) which is established through second messengers such as calcium and cyclic nucleotides. The sensory function of axons is dependent on cues from the extracellular matrix which can be either attractive or repulsive, thus helping to guide the axon away from certain paths and attracting them to their proper target destinations. Attractive cues inhibit retrograde flow of the actin filaments and promote their assembly whereas repulsive cues have the exact opposite effect. Actin stabilizing proteins are also involved and are essential for continued protrusion of filopodia and lamellipodia in the presence of attractive cues, while actin destabilizing proteins are involved in the presence of a repulsive cue.
A similar process is involved with microtubules. In the presence of an attractive cue on one side of the growth cone, specific microtubules are targeted on that side by microtubule stabilizing proteins, resulting in growth cone turning in the direction of the positive stimulus. With repulsive cues, the opposite is true: microtubule stabilization is favored on the opposite side of the growth cone as the negative stimulus resulting in the growth cone turning away from the repellent. This process coupled with actin-associated processes result in the overall directed growth of an axon.
Growth cone receptors detect the presence of axon guidance molecules such as Netrin, Slit, Ephrins, and Semaphorins. It has more recently been shown that cell fate determinants such as Wnt or Shh can also act as guidance cues. Quite interestingly, the same guidance cue can act as an attractant or a repellent, depending on context. A prime example of this is Netrin-1, which signals attraction through the DCC receptor and repulsion through the Unc-5 receptor. Furthermore, it has been discovered that these same molecules are involved in guiding vessel growth. Axon guidance directs the initial wiring of the nervous system and is also important in axonal regeneration following an injury.

References[edit]

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  • Kandel ER, Schwartz JH, Jessell TM 2000. Principles of Neural Science, 4th ed. pp.1070–1074. McGraw-Hill, New York. ISBN 0-8385-7701-6
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