During neural development, highly motile structures on the developing neurites, called growth cones, are guided by signals from the extracellular envrionment. Guidance cues come in many different forms, from diffusible extracellular proteins and lipid factors, to extracellular matrix proteins and/or carbohydrates located on the cell substrate. They may also originate from membrane of adjacent cells.
To be considered a guidance cue, the molecule or protein must meet the following criteria:
In most cases, there are multiple receptors for each guidance cue; however, it is widely known that the final outcome resulting from guidance cue-induced events is dependent upon the cell type, the duration the signal is ‘on’, the state of the cell (e.g. differentiation state, cell cycle stage, etc), and the concentration of the guidance cue.
The guidance signals for neuronal growth cone movement are among the most widely studied  (reviewed in ). The generation of a signal concentration gradient allows cells to use a directional sensing program to generate “front”-specific responses at the region where the signal is the highest and “rear” specific responses where the signal is the lowest. The activation of localized signal transduction events results in positional cues that promote the relocalization of certain proteins to the front or back of the cell. Subsequent cytoskeletal restructuring redirects the migration of the growth cone (reviewed in ). The final response to many guidance signals can be affected by the level of both intra- and extracellular calcium .
Most known attractive signals act as chemoattractants, often generating the formation of adhesion molecules within the growth cone to promote selective extension of the filopodia towards the cue, whilst ensuring the formation of filopodia or lamellipodia is decreased in other directions. This occurs through a decreased retrograde actin flow in the direction of the contact [20, 21]. Such attractive cues are propagated by the Rho family of GTPases (e.g. Rac1, Cdc42 and RhoA) following ligand-induced receptor binding to the cytoskeleton (reviewed in ). Attractive cues such as brain-derived neurotrophic factor (BDNF) activate Cdc42 and Rac , and Rac1 activity in turn influences the initial formation of integrin-dependent adhesions in the growth cone. In this case, further adhesion stabilization and continued migration requires proper coordination between Rac1 and RhoA GTPase activity . Local signals may also redirect the signal through other recruited components .
In the case of repulsive signals, the actin assembly module is suggested to rapidly terminate growth in synchrony with the release of linkages between the cytoskeleton and the membrane . Growth cone collapse is a key example of how neural cells respond to repulsive cues by altering actin filament dynamics. Growth cone collapse involves numerous signaling pathways including Rho GTPases , ADF , and kinases .
A model for filopodia collapse in growth cones was created using the repulsive signal, semaphorin IIIA (SemaIIIA; collapsin-1). Another example is reelin, a large secretory protein that inhibits filopodia formation through a pathway involving Fyn or Src kinase-mediated phosphorylation and inactivation of mDab1 (mouse Disabled homologue 1) . Phosphorylation of mDab1 reduces the activation of N-WASP and Arp2/3-dependent actin polymerization .
It is not always clear whether a guidance signal provides information for both directional and temporal movement. However, they can be integrated temporally and/or spatially:
Lamellipodial and filopodial protrusions at the leading edge of migrating cells and neural growth cones are exposed to a number of guidance cues simultaneously and the proper integrated response is necessary for proper cell/growth cone guidance and maintenance. In certain cases, prior exposure to one signal initiates cytoplasmic events that prevent or ‘desensitize’ the cell to subsequent guidance cues (reviewed in ).
Cyclic nucleotides can regulate growth cone behaviours by converting repulsive signals into attractive signals through the activation of cyclic guanosine 3′,5′-monophosphate (cGMP) and cyclic adenosine 3′,5′-monophosphate (cAMP) signaling pathways, respectively (reviewed in ). For example, the small molecule netrin, induces attractive or repulsive signals depending on the type of receptors expressed and on the intracellular level of cAMP .
Other guidance cues function as attractants for one navigational event and a repellent for another (reviewed in ); similarly, the signal molecules that are activated by these guidance cues frequently antagonize each others’ activity, emphasizing the importance of proper coordination between these signaling components . Not surprisingly, mutation(s) in the signaling pathways that are used by guidance cues frequently results in aberrant navigation (reviewed in ).
Stimulating filopodia formation A secreted guidance cue which stimulates filopodia formation in growth cones. Two examples of secreted neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), mediate retinal ganglion cell axon filopodia  and sprouting of axonal filopodia, respectively . Neurotrophins bind Trk receptors and the p75 neurotrophin receptor (p75NTR) to regulate both filopodia assembly and length . Neurotrophins stimulate filopodial assembly by influencing actin-dependent nucleation, polymerization, and motility through PI-3 kinase-dependent signal transduction pathways .
Inducing filopodia collapse in chick and its mammalian orthologue, semaphorin III (SemaIII) are a few of the most extensively studied proteins that cause repulsion or collapse of growth cones in particular neuronal classes. Members of this group are secreted and transmembrane proteins; they bind to neurophilin receptors to stimulate repulsion (reviewed in ). SemaIIIA causes termination of protrusive activity and growth cone collapse  through activation of Rac1  and decreased phosphorylation of the ezrin–radixin–moesin (ERM)family of F-actin binding proteins . Phosphorylation of ERM proteins activates the F-actin binding domain and regulates filopodia assembly/protrusion by linking filopodial membranes with F-actin (reviewed in ). Inactivation of the phosphoinositide 3-kinase (PI3K) signal pathway by SemaIIIA may also be linked to reduced ERM protein activity and growth cone collapse .
Membrane-bound ephrins can be divided into two main classes, determined by sequence homology, which reflect the means by which they attach to the membrane. Ephrin As bind to the membrane via glycosylphosphatidylinositol (GPI) anchors while ephrin Bs traverse the membrane via their transmembrane domain and contain a short cytoplasmic tail .
In general ephrin As bind Eph A receptors and ephrin Bs bind Eph B receptors [Gale] and in both instances this facilitates bidirectional signaling, though reverse signaling in ephrin A-Eph A complexes is poorly understood. Both ephrin As and Bs contain a well conserved extracellular domain that binds Eph receptors. Ephrin Bs additionally contain conserved tyrosine phosphorylation sites and a PDZ domain in their cytoplasmic tails that facilitate intracellular signaling (reviewed in ).
The primary role of ephrin-Eph receptor signaling is to regulate cellular patterning, e.g. guiding neuronal growth cones to the appropriate target. A significant body of work has elucidated the role of ephrins in neural development (as reviewed in ). For example; ephrin Bs are of particular importance in patterning of the hindbrain – a distinctly segmented structure within the brain. Ephrins Bs and their cognate Eph receptors are expressed in complementary domains, such that mixing of neuronal cells is prevented by repulsive cues resulting from the interaction of ephrin Bs and Eph receptors at the interfaces between two adjacent domains .
Other processes highly dependent on cell-cell contacts that involve ephrins include the maturation of thymocytes into T-cells of the immune system, the release of insulin from pancreatic beta cells, osteoblast differentiation and segregation of dividing and differentiated cells of the intestinal epithelium. Under pathological conditions, the dysregulation of Eph-ephrin expression has been implicated in cancer and in the development of tumor vasculature (tumour angiogenesis) (reviewed in ).