Tropic cues involved in growth cone guidance

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 motif important 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. 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 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 the Neuropilin 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.