InteractiveFly: GeneBrief

Plexin A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Before a closer look at Plexin A, the subject of this overview, it might be useful to look at the Plexin family of proteins in terms of its relationship to two other protein families: Semaphorins and Neuropilins.

To date, searching the genomes of the nematode C. elegansand the fruit fly Drosophila has not revealed homologs for Neuropilins, the receptors (or receptor subunits) for class III Semaphorins (He, 1997 and Kolodkin, 1997). Nematodes and fruit flies also appear to lack class III Semaphorins; worms have classes I and II, while flies have classes I, II, and V. Both these invertebrate genomes also appear to lack MET tyrosine kinase receptor homologs (serving as receptors for semaphorins in vertebrates), but they do contain genes encoding Plexins. With the lack of Neuropilins and MET tyrosine kinase receptors in worms and flies, it is suggested that Plexins are the oldest Semaphorin-binding proteins. It may be that during chordate evolution, a new class of Semaphorins (type III) evolved, and with them, a new class of Semaphorin receptors (the Neuropilins) (Winberg, 1998 and references therein).

What proteins serve as receptors for Semaphorins? Neuropilin-1, originally discovered in Xenopus, is expressed by specific layers in the optic tectum. Though it can mediate homotypic adhesion, recent studies have revealed that Neuropilin-1 also binds with high affinity many class III Semaphorins, including Sema III, Sema E, and Sema IV. Clues as to the nature of additional Semaphorin receptors come from examination of viral Semaphorins. The genomes of several viruses, including the poxviruses vaccinia and variola (Kolodkin, 1993) and a herpesvirus (Ensser, 1995), harbor open reading frames encoding secreted Semaphorins that might modulate immune function. One such viral Semaphorin, vaccinia A39R, was used to affinity purify a novel cellular receptor from a human B cell line (Comeau, 1998). This virus-encoded Semaphorin protein receptor (VESPR) is a novel transmembrane protein that is a divergent member of the Plexin family. VESPR binds A39R with high affinity and herpesvirus AHV Sema with lower affinity. Human Sema K1 is a class VII Semaphorin that may be the cellular counterpart of AHV Sema; Sema K1 does not bind Neuropilin (Winberg, 1998 and references therein).

Like Neuropilins, Plexins were identified through a screen for antigens expressed in the optic tectum (Takagi, 1987). Xenopus Plexin-1 has been shown to contain an extracellular domain related to the c-met protooncogene and to mediate calcium-dependent homophilic binding (Ohta, 1995). Neuropilin-1 and Plexin-1 are expressed on different groups of olfactory axons that independently segregate, leading to the speculation that these two proteins help organize axon projections (Satoda, 1995). Plexin-1 was subsequently cloned in mice and is the prototype of a family of proteins (Kameyama, 1996). Plexins in human were cloned independently, based on their homology to the ectodomain of the oncogene MET (Maestrini, 1996). Called the SEX family of genes, several members (SEX, OCT, and NOV) are expressed predominantly in the brain, giving rise to the suggestion that they represent novel neuronal receptors for unknown ligands (Winberg, 1998 and references therein)

Given that a divergent member of the Plexin/SEX family is a receptor for the viral Semaphorin A39R and that most Plexins are expressed in the developing nervous system, Plexins have been hypothesized to serve as neuronal Semaphorin receptors. It was thought that they could function to control axon guidance. To investigate this possibility, the genes encoding two Plexins in Drosophila were cloned and characterized. Genetic and biochemical evidence support the idea that Plexin A (PlexA) in Drosophila is a receptor for the transmembrane class I Semaphorins Sema 1a and 1b. PlexA has been shown to control important aspects of axon guidance. Because there are two class I Semaphorins and two class II Semaphorins in Drosophila, the original Sema I has been renamed Sema 1a; the new class I Semaphorin is Sema 1b; the original Sema II is now Sema 2a, and the new class II Semaphorin is Sema 2b. Previous studies (Yu, 1998) have shown that mutations in Sema1a display specific defects in motor and CNS axon guidance. Mutations in PlexA display the same phenotypes and the two loci interact genetically in a fashion consistent with their functioning in the same signaling pathway. Along with selective binding results, these data show that Plexins do indeed function as neuronal Semaphorin receptors and that Plexins control important aspects of axon guidance (Winberg, 1998).

What about the neuropilins? It has been observed that the main semaphorin-binding domain of neuropilins (CUB domain) is not required for the interaction with plexins, as indicated by the association of the relevant neuropilin-2 deletion construct with plexin-B1. Thus the existence of a ternary complex is envisioned, where neuropilins use two distinct protein modules to form a bridge between the sema domain of semaphorins and the sema domain of plexins. Taken together, these findings raise the possibility that plexins are the long-sought functional partners of neuropilins required for transducing signals mediated by class 3 semaphorins. In flies, which lack both neuropilins and class 3 semaphorins, it is notable that Drosophila PlexA appears sufficient as a functional receptor for Sema-1a, a transmembrane class 1 semaphorin (Winberg, 1998). In further support of the hypothesis that plexins are functional coreceptors for secreted semaphorins, it has been shown that a truncated human plexin-A1 construct expressed in Xenopus spinal neurons abolishes repulsive responses to Sema3A without markedly affecting attractive responses to netrin-1. Similarly, expression of a dominant-negative plexin-A1 in sensory neurons blocks Sema3A-induced growth cone collapse, as reported independently by Takahashi (1999). Therefore, neuropilins serve along with plexins as co-receptors and receptors for Semaphorins (Tamagnone, 1999).

Sema 1a is expressed by neurons and is required for appropriate defasciculation. Loss-of-function analysis for this gene does not indicate whether Sema 1a functions as a ligand or as a receptor. However, misexpressing Sema 1a on muscles repels motor axons, demonstrating that Sema 1a is able to act as a target-derived repulsion cue (Yu, 1998). If PlexA is the receptor for Sema 1a, then reducing PlexA expression levels in the presence of ectopic Sema 1a ligand should suppress the severity of the gain-of-function repulsion phenotypes. Two different GAL4 enhancer trap lines were used to misexpress Sema 1a on muscle subsets. The first, H94-GAL4, is highly expressed by muscle fibers 6 and 13, and moderately by muscle 12 (genetic rescue data suggest that it is also expressed by some motor neurons at a very low level, although this has never been directly visualized. Using H94-GAL4 to drive UAS-Sema1a in these muscles disrupts their innervation by ISNb axons, with the strongest effect seen at muscle 13. A second line, F63-GAL4, was used to drive UAS-Sema1a specifically in muscles 6 and 7, thereby inhibiting innervation at the muscle 6/7 cleft (F63 is not expressed by motor neurons). For both GAL4 lines, the inhibitory effect of muscle-derived Sema 1a is suppressed in PlexA Df heterozygotes. This dominant suppression of the Sema1a gain-of-function suggests that neuronally expressed PlexA acts downstream of Sema 1a in mediating repulsion (Winberg, 1998).

Ectopic expression also provided a means to test for interactions between PlexA and Sema 1b, another Drosophila protein similar to Sema 1a (Yu, 1997). H94-GAL4 and F63-GAL4 were used to test whether Sema 1b can also act as a muscle-derived repellent. Sema 1b was found to be as capable as equally capable Sema 1a in repelling motor axons. Likewise, reducing the gene dose of PlexA suppresses to a similar extent the guidance defects caused by misexpression of Sema 1b. Based on the similarity of structure and sequence, as well as parallel gain-of-function phenotypes and suppression, it is proposed that Sema 1b may serve as an additional ligand for PlexA (Winberg, 1998).

Given the model that PlexA mediates repulsive guidance and thus drives defasciculation, it was asked whether removal of the major motor axon cell adhesion molecule, Fasciclin II, would genetically suppress PlexA mutant phenotypes. In agreement with the model, all of the ISNb motor axon PlexA phenotypes (but not the SNa mutant phenotypes) and the CNS PlexA phenotypes are partially suppressed when one copy of the FasII gene is removed (Winberg, 1998).

Defects arising from overexpression of PlexA were also examined. Driving high levels of UAS-PlexA in all neurons using elav-GAL4 leads to axon guidance phenotypes in all parts of the motor projection and also within the CNS. In most cases, PlexA overexpression results in phenotypes that are the opposite of those seen in the PlexA loss-of-function. For example, in 66% of segments, PlexA overexpression caused the dorsal SNa to split prematurely into multiple projections. Similarly, the dorsal extension of the ISN defasciculated inappropriately in 25% of segments. The transverse nerve, which made exuberant contacts onto ventral muscles in the deficiency embryos, stalled in 26% of segments with PlexA overexpression, resulting in the two halves of the nerve failing to meet. These defects are readily interpreted as resulting from increased sensitivity to repulsive cues (Winberg, 1998).

Genetic analysis provides strong evidence that PlexA is a necessary component of the Sema 1a signaling pathway and is likely to function as a Sema 1a receptor. Moreover, it suggests that PlexA may have additional ligands, including Sema 1b. These possibilities were tested in a heterologous expression system using alkaline phosphatase (AP) fusion proteins in binding assays on membranes from COS cells transiently transfected with a full-length PlexA construct. Both AP-Sema 1a and AP-Sema 1b bind to PlexA-expressing membranes at significantly higher levels than to mock-transfected membranes or to membranes containing another Drosophila repulsive axon guidance receptor, Robo 1. No specific binding occurs between PlexA and other Semaphorins, including Sema III-AP, AP-Sema E, and AP-Sema B, nor between PlexA and AP-Beaten path. It was also asked whether Sema-Plexin binding, like Plexin homophilic interaction in Xenopus, requires the presence of divalent cations. Binding is eliminated by the inclusion of chelating agent in the ligand supernatant, or by the omission of divalent cations from the wash buffer. Binding is preserved in the presence of either Mg2+ or Ca2+. In contrast, binding of Semaphorins to Neuropilins does not appear to require calcium or magnesium (Winberg, 1998).

Comeau (1998) noted that VESPR and other Plexins share a ~100 amino acid region of homology with Semaphorins near the C terminus of the Sema domain. Upon further sequence analysis, Plexins and their relatives, the MET-related tyrosine kinase receptors (including MET, RON, and SEA), all are found to contain a complete ~500 amino acid Sema domain near the N terminus of their ectodomains. Thus, Plexins may be considered as large transmembrane Semaphorins. The CLUSTAL algorithm was used to examine the relationships among the Sema domains of Plexins, Semaphorins, and MET-related receptors. Each of the three groups of proteins clusters separately. The structure of the tree suggests a phylogenetic model in which Plexins may have been the ancestral molecules from which Semaphorins and MET-related receptors evolved; from this perspective, Semaphorins may be considered as specialized Plexins (Winberg, 1998).

The data are consistent with a model in which Semaphorins are ligands and Plexins are receptors mediating repulsive axon guidance. Such a model may capture only part of the complexity of how these molecules function. The finding that Plexins can bind Semaphorins, combined with previous studies showing that Plexins can bind Plexins (Ohta, 1995), raises questions as to whether some Semaphorins might bind one another and whether class I Semaphorins might also function as receptors. In the immune system, CD100, a class IV Semaphorin, promotes B cell aggregation and also plays a role in T cell activation (Hall, 1996 and Herold, 1996). Some of its functions are consistent with CD100 functioning as a receptor as well as a ligand (Winberg, 1998).

Function of the Drosophila receptor guanylyl cyclase Gyc76C in PlexA-mediated motor axon guidance

The second messengers cAMP and cGMP modulate attraction and repulsion mediated by neuronal guidance cues. The Drosophila receptor guanylyl cyclase Gyc76C genetically interacts with Semaphorin 1a (Sema-1a) and physically associates with the Sema-1a receptor plexin A (PlexA). PlexA regulates Gyc76C catalytic activity in vitro, and each distinct Gyc76C protein domain is crucial for regulating Gyc76C activity in vitro and motor axon guidance in vivo. The cytosolic protein dGIPC interacts with Gyc76C and facilitates Sema-1a-PlexA/Gyc76C-mediated motor axon guidance. These findings provide an in vivo link between semaphorin-mediated repulsive axon guidance and alteration of intracellular neuronal cGMP levels (Chak, 2013).

Both membrane-associated and secreted neuronal guidance cues can attract or repel axons and dendrites during neural development, and several families of guidance cues and receptors perform these functions. Modulation of guidance cue activities through intracellular signaling components determines how extrinsic factors are interpreted by extending neuronal processes during development. For example, growth cone turning experiments in vitro demonstrate that attraction mediated by the guidance cue netrin-1 can be converted to repulsion by lowering intracellular cAMP (Ming, 1997), whereas repulsion mediated by the guidance cue Semaphorin 3A (Sema-3A) can be converted to attraction by increasing intracellular cGMP (Song, 1998). Elevated cAMP in cultured DRG neurons neutralizes Sema-3A growth cone collapse, whereas elevated cGMP potentiates it (Dontchev, 2002). The ratio of cAMP to cGMP can determine the sign of a growth cone steering response (Nishiyama, 2003), and Sema-3A induces cGMP production in neuronal growth cones, activating of cGMP-gated calcium channels (CNGCs), Ca2+ influx and repulsion (Togashi, 2008). cAMP and cGMP regulate kinases and phosphodiesterases to direct formation of axons or dendrites in cultured hippocampal neurons (Shelly, 2010). Therefore, coordination of cAMP and cGMP signaling regulates cellular responses to different stimuli in the neurons. Guanylyl cyclases (GCs) include soluble and transmembrane proteins that catalyze the conversion of GTP to cGMP, and they regulate a wide range of diverse cellular and physiological processes (Davies, 2006), including axonal and dendritic guidance (Polleux, 2000; Seidel, 2000; Gibbs, 2001; Nishiyama, 2003). The mammalian receptor guanylyl cyclase GC-B and cGMP-dependent kinase I (cGKI) are essential for proper sensory axon afferent guidance into the CNS, and C-type natriuretic peptide is the GC-B ligand that is crucial for murine sensory axon branching, axon outgrowth and axon attraction (Schmidt, 2009; Zhao, 2009). Yet, how GCs are linked to axon guidance signaling to alter intracellular cGMP levels and modulate growth cone responses in vivo is unclear (Chak, 2013).

The Drosophila transmembrane semaphorin Sema-1a binds to the plexin A (PlexA) receptor to mediate axon-axon repulsion and to control axonal fasciculation in embryonic central and peripheral nervous systems (CNS and PNS). The Drosophila receptor GC Gyc76C is required in motoneurons for Sema-1a-PlexA-mediated axon guidance and is dependent on the integrity of the Gyc76C catalytic cyclase domain (Ayoob, 2004). This study investigated connections between Gyc76C and Sema-1a-PlexA-mediated axon guidance. The findings support the idea that Gyc76C-generated cGMP within neuronal growth cones facilitates axonal repulsion mediated by Sema-1a and PlexA, allowing for the establishment of Drosophila embryonic neuromuscular connectivity (Chak, 2013).

Gyc76C-Sema-1a gain-of-function genetic interactions observed in this study are consistent with previous observations showing that Gyc76C loss and gain of function modifies aberrant CNS midline crossing by FasII+ longitudinal axons in a PlexA gain-of-function genetic background (Ayoob, 2004). Furthermore, robust physical interactions were observed between Gyc76C and PlexA both in vitro and in vivo, raising the possibility that PlexA regulates Gyc76C-mediated signaling. Co-expressing PlexA at high levels in vitro augments cGMP levels produced by Gyc76C. Future work will establish whether extracellular, intracellular, or both, types of protein-protein associations between Gyc76C and PlexA are essential for regulating Gyc76C enzymatic activity (Chak, 2013).

Gyc76C structure-function analyses are consistent with the idea that PlexA binds to the extracellular and intracellular regions of Gyc76C to relieve inhibitory effects on GC activity from of Gyc76C intramolecular interactions, increasing cGMP levels within extending motor axon growth cones and affecting growth cone guidance. This is reminiscent of Sema-3A bath application increasing intracellular cGMP levels in Xenopus spinal neurons in vitro, and the results suggest that intracellular cGMP produced by Gyc76C is required for Sema-1a-mediated repulsion. However, it is possible that signaling by intracellular cGMP is coupled with intracellular cAMP in Sema-1a-mediated repulsive guidance events (Nishiyama, 2003), and future work will determine whether varying the cAMP-to-cGMP ratio modulates Sema-1a-mediated repulsion, or converts it to attraction. Bath application of Sema-1a did not affect Gyc76C-PlexA physical associations or Gyc76C-PlexA-mediated cGMP, suggesting that Sema-1a-dependent regulation of intracellular cGMP levels could involve ligand-gated, dynamic, spatiotemporal regulation of GC activity; visualizing this signaling event will require real-time imaging of cGMP during repulsive growth cone steering (Chak, 2013).

The small GTPase Rac and its downstream effector p21 activated kinase (PAK) can regulate receptor GCs to raise cellular cGMP levels in fibroblasts in vitro, and the kinase domain of PAK interacts with the cyclase domain of receptor GC-E (Guo, 2007; Guo, 2010); PAK, therefore, may associate with Gyc76C and regulate this receptor GC during axon pathfinding (Chak, 2013).

The deletion of the Gyc76C PDZ-binding motif (PBM) strongly suppresses cGMP production by FL Gyc76C, and Gyc76C variants lacking the PBM motif exhibit low cell-surface expression levels, suggesting dGIPC regulates Gyc76C cell-surface localization. PDZ-containing proteins could form a protein scaffold required for plasma membrane localization of the Sema-1a-PlexA/Gyc76C signaling complex, and the PDZ domain-containing dGIPC protein was found to interact with Gyc76C. In vertebrates, GIPC regulates protein trafficking, subcellular localization and various signaling events. Gyc76C cell-surface localization is enhanced in vitro in the presence of dGIPC, and this may serve to regulate Gyc76C-mediated signaling. dGIPC genetic analyses show that dGIPC plays a neuronal role in motor axon guidance, and this likely occurs through interactions that modulate Gyc76C-mediated cGMP signaling. Mammalian GIPC forms dimers and multimeric complexes. dGIPC may be a part of a molecular scaffold that couples Gyc76C to cell membrane trafficking machinery or anchors Gyc76C to the plasma membrane. Alternatively, dGIPC may be essential for activating or transducing Gyc76C-mediated cGMP signaling in axon guidance events. Future genetic and biochemical analyses will reveal the downstream signaling components that respond to changes in cGMP levels in vivo and direct discrete neuronal growth cone steering responses (Chak, 2013).

Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells

Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces-one composed of the atypical cadherin Fat2 and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptorPlexin A. This study shows that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at cells' leading edges, but Lar and Semaphorin5c play little role in Fat2's localization. Fat2 is also more stable at interfaces than Lar and Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. It is proposed that Fat2 serves as the organizer this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells (Williams, 2023).

Epithelial cells migrate collectively during animal development, wound healing, intestinal turnover and cancer metastasis. To do so, they must polarize within the epithelial plane at both the individual and tissue scales. At the individual scale, cells polarize along a leading-trailing axis. Protrusion and adhesion formation are biased to the leading edges of cells, and contractility and adhesion removal to their trailing edges, much as in cells migrating solo. At the tissue scale, cells throughout the epithelium are polarized, such that their leading edges preferentially point in the direction of migration, and trailing edges in the opposite direction, a form of planar cell polarity. At the intersection of these scales are the cell–cell interfaces that link the trailing edge of one cell to the leading edge of the cell behind. These leading-trailing interfaces can act as sites of biochemical or mechano-chemical signaling that polarize motility behaviors across the interface. However, little is known about how signaling proteins become organized along interfaces to accomplish this feat (Williams, 2023).

The rotational migration of the follicle cells in Drosophila melanogaster has proven to be a fruitful system for identifying signaling mechanisms that coordinate epithelial cell movements. Follicle cells are somatic cells of the egg chamber, the multicellular structure within the ovary that gives rise to an egg. They form a continuous monolayer epithelium around a central cluster of germ cells, and they are surrounded in turn by a basement membrane extracellular matrix that encapsulates the entire egg chamber. The apical surfaces of the follicle cells adhere to the germ cells, and their basal surfaces adhere to and crawl along the basement membrane. Migration in this topologically closed configuration causes the entire egg chamber to rotate within the stationary basement membrane. This motion changes the structure of the basement membrane, ultimately helping give the egg its elongated shape. Follicle cell migration requires WAVE complex-dependent lamellipodia, which are polarized to the leading edge of each cell and planar-polarized across the epithelium. Polarity emerges tissue-autonomously, without input from extrinsic directional cues. This simplifying feature allows more easy isolation od the contribution of within-group coordination to collective migration. It likely also makes these cells particularly reliant on such coordination for movement (Williams, 2023).

Two biochemical signaling modules operate at leading-trailing interfaces, where they coordinate the migratory behaviors of neighboring follicle cells. The first module is composed of the atypical cadherin Fat2 (also known as Kugelei) and the receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar). Fat2 is enriched along the trailing edge of each cell, where it acts in trans to concentrate Lar and the WAVE complex across the cell–cell interface, at the leading edge of the cell behind. Lar also contributes to WAVE complex localization, but not as strongly as Fat2, implying the existence of additional unidentified Fat2 effectors. Together, these proteins restrict cell protrusive activity to a single leading-edge domain and orient the protrusions from all the cells in a uniform direction across the tissue. The second module is composed of a transmembrane semaphorin (ligand) and plexin (receptor) pair, Semaphorin 5c (Sema5c) and Plexin A (PlexA), which are enriched at leading and trailing edges respectively. In other contexts, semaphorin–plexin signaling can lower integrin-based adhesion and/or inhibit protrusivity on the plexin-containing cell side. Similarly, overexpression of Sema5c in one follicle cell reduces the protrusivity of its neighbors in a PlexA-dependent manner. This led to the model that Sema5c signals through PlexA to maintain a non-protrusive state at the trailing edges of cells (Williams, 2023).

Despite their distinct depletion and overexpression phenotypes, several lines of evidence suggest that the Fat2–Lar and Sema5c–PlexA modules function within one interface-polarizing signaling system. A series of pairwise comparisons show that Fat2, Lar and Sema5c all colocalize with the WAVE complex in interface-spanning puncta that sit at the tips of filopodia within a broader lamellipodium. For reasons that are not yet clear, PlexA only rarely colocalizes with the other proteins. Loss of Lar also reduces the enrichment of Sema5c at leading edges, implying functional interaction between the two modules in addition to their shared spatial organization. This study investigated the hierarchy of interactions between Fat2, Lar, and Sema5c by which they form interface-spanning puncta, and asked how the three proteins work together to promote collective migration (Williams, 2023).

Fat2 was found to form the core of both the Lar and Sema5c-containing signaling modules, concentrating Sema5c at leading edges in trans as it was previously shown to do for Lar. Conversely, Lar and Sema5c play little or no role in the localization of Fat2. Using fluorescence recovery after photobleaching (FRAP) and acute inhibition experiments, it was shown that Fat2 resides more stably at trailing edges than do Lar or Sema5c at leading edges, and that Fat2 is likely continuously required to maintain the enrichment of Lar and Sema5c at leading edges in the face of their ongoing turnover. It was further found that Lar and Sema5c act in parallel to promote collective migration. From these data, it is proposed that Fat2 acts as a central organizer of the follicle cell interface-polarizing signaling system, serving to couple and polarize the distributions of multiple effectors that together align the motility machinery of neighboring cells (Williams, 2023).

The follicle cells use biochemical signaling across their leading-trailing interfaces to polarize their migration machinery at interface, cell and tissue scales. This study has shown that Fat2 is a central organizer of this signaling system. Fat2 acts at the trailing edge of each cell to concentrate both Lar and Sema5c at the leading edge of the cell behind. By contrast, Lar and Sema5c play at most minor roles in the localization of Fat2. In this way, Fat2 coordinates the activities of two effector proteins with distinct functions, allowing them to work synergistically to promote highly persistent collective migration (Williams, 2023).

One defining feature of this Fat2-based signaling system is that most of the component proteins colocalize in interface-spanning puncta. Cadherins often self-organize into clusters, making it likely that this punctate organization stems from Fat2. However, whether Fat2 concentrates Lar and Sema5c in the puncta through direct binding or through intermediary proteins is unknown. An ectopic Fat2 pool did not cause redistribution of Lar, suggesting that additional inputs also contribute to the localization of Lar. Also it is not known known how the receptor for Sema5c, PlexA, fits into this model. Like Fat2, PlexA is enriched at the trailing edges of cells and helps to localize Sema5c, and yet antibody staining indicates that PlexA only partially colocalizes with the Fat2-based puncta. The biggest open question is how Fat2 becomes localized to the trailing edge, as this appears to be the key event that polarizes the entire signaling system. Mechanical feedback from collective migration itself is required for the polarization of Fat2 to trailing edges, but the nature of this feedback, and whether Fat2 has a trans binding partner that further stabilizes its localization, will be important areas for future investigation (Williams, 2023).

This study includes the first measurements of the dynamics of planar signaling proteins in follicle cells, which is an important step towards understanding their polarization mechanism. Using FRAP, it was found that Fat2 is a more stable resident of the leading-trailing interface-spanning puncta than are Lar or Sema5c, consistent with its more central role in maintaining these structures. Based on these FRAP data, as well as the rapid redistribution of Lar and Sema5c upon extracellular Ca2+ chelation, it is hypothesized that Fat2 maintains the leading edge enrichment of Lar and Sema5c by slowing their turnover within the puncta, thereby concentrating them at the leading edge. However, the continuous requirement of Fat2 for the maintenance of polarization of Lar and Sema5c awaits confirmation with a more specific method of acute Fat2 inhibition, as extracellular Ca2+ chelation is a blunt tool, and it is possible that Fat2-independent effects, such as the disruption of another cadherin, contributed to the rapid localization changes of Lar and Sema5c. Further comparison of the dynamics of Lar and Sema5c with and without Fat2 will also be needed to determine whether Fat2 concentrates them through local stabilization (for example through direct or indirect binding) or by a different mechanism, such as increasing their rate of arrival at leading edges (Williams, 2023).

This work also sheds light on how Lar and Sema5c work together to promote collective migration. Previously work has shown that loss of either protein alone impairs migration but does not stop it. By contrast, it is now found that removing both proteins together fully blocks migration in a way that is indistinguishable from loss of Fat2. These data suggest that once Fat2 concentrates Lar and Sema5c to the leading edges of cells, they then act in parallel to promote collective migration, likely by polarizing distinct aspects of the migration machinery (Williams, 2023).

What aspects of the migration machinery do Lar and Sema5c each control? In the case of Lar, it seems to be part of the bridge between Fat2 and WAVE complex-dependent protrusions — both Fat2 and Lar increase protrusive F-actin enrichment at leading edges (Fat2 in trans and Lar in cis), and both also help polarize protrusions in the direction of migration. In addition, Lar acts in trans to promote retraction of the trailing edge of the cell ahead, but the mechanistic basis for this trans function and the degree to which it is separable from the cis function of Lar, remain undetermined. In the case of Sema5c, cell-scale loss-of-function phenotypes have proven more elusive, but enrichment of PlexA at trailing edges and the ability of overexpressed Sema5c to suppress protrusion in trans both point to the trailing edge as the likely site of regulation (Williams, 2023).

By positioning both Lar and Sema5c, Fat2 integrates two features of interface signaling systems known to operate in other collectively migrating cell types but not yet seen together. The first feature is the use of a trailing edge-associated mechanical cue to orient protrusions in the cell behind. Mechanical localization of Fat2 to trailing edges polarizes protrusions in the following cell, in part by localizing Lar. The second feature is the use of contact inhibition of locomotion, which causes cells to polarize away from one another by suppressing protrusion and/or increasing contractility at the point of contact. By localizing Lar and Sema5c to leading edges, Fat2 positions them to enforce trailing edge behavior at the contacting edge of the cell ahead. Therefore, Fat2 translates a mechanical cue into bi-directional signaling across leading-trailing interfaces to coordinate cell migratory behaviors for collective migration (Williams, 2023).

REGULATION

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. This study, using extensive mutagenesis of expression plasmids and RNAi screening, revealed that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (approximately 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, it was found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes (Kramer, 2015).

It was long assumed that eukaryotic pre-mRNAs are always canonically spliced to generate a linear mRNA that is subsequently translated to produce a protein. However, it is now becoming increasingly clear that many genes can be noncanonically spliced to produce circular RNAs with covalently linked ends. These transcripts are almost exclusively derived from exons, accumulate in the cytoplasm, and are thought to be products of alternative splicing events known as 'backsplicing.' In contrast to canonical splicing, which joins the exons in a linear order (joining exon 1 to exon 2 to exon 3, etc.), backsplicing joins a splice donor to an upstream splice acceptor (e.g., joining the 3' end of exon 2 to the 5' end of exon 2). A handful of RNAs generated in this manner were identified in the 1990s, and recent deep sequencing studies have expanded this observation to thousands of circular RNAs expressed across eukaryotes, including humans, Caenorhabditis elegans, Drosophila (Salzman. 2013; Ashwal-Fluss, 2014; Westholm, 2014), Schizosaccharomyces pombe, and plants. Perhaps surprisingly, for some genes, the abundance of the circular RNA exceeds that of the associated linear mRNA by a factor of 10, suggesting that the major function of some protein-coding genes may be to generate circular RNAs (Kramer, 2015).

Most exons in eukaryotic genomes have splicing signals at both ends and theoretically can circularize. However, only certain exons are observed in circular RNAs, and these backsplicing events often occur in a tissue-specific manner. This suggests that circular RNA biogenesis is tightly regulated. As splicing generally occurs cotranscriptionally, most introns, along with their upstream splice acceptors (which are needed for backsplicing), are rapidly removed. Therefore, for circular RNAs to be produced, canonical splicing likely must occur more slowly around these exons, and/or exon skipping events may be coupled to circular RNA biogenesis. In the latter, the circular RNA is derived from an exon-containing lariat, allowing a pre-mRNA to yield both a linear mRNA and a circular RNA comprised of the skipped exons (Kramer, 2015).

There is little known about the splicing factors that regulate these events. In some cases, the Muscleblind (Mbl) and Quaking proteins appear to facilitate backsplicing by bridging between two introns and causing the splice sites from the intervening exons to be brought into close proximity (Ashwal-Fluss, 2014; Conn, 2015). For example, circular RNA production from the Drosophila mbl gene is triggered when the Mbl splicing factor binds to its own introns (Ashwal-Fluss, 2014). However, in humans, mice, and C. elegans, the predominant determinants of whether a pre-mRNA is subjected to backsplicing are intronic repetitive elements, such as sequences derived from transposons. Almost 90% of human circular RNAs have complementary Alu elements in their flanking introns, and, analogous to the protein-bridging mechanism, base-pairing between complementary sequences allows the intervening splice sites to be brought close together. Interestingly, repeats <40 nucleotides (nt) can drive circular RNA production in human cells, but it is clear that more than simple thermodynamics regulates circularization. For example, base-pairing interactions can be disrupted by ADAR (adenosine deaminase acting on RNA), which converts adenosines in double-stranded regions to inosines. In addition, most mammalian pre-mRNAs contain multiple intronic repeats, allowing distinct circular (or linear) RNAs to be produced depending on which repeats base-pair to one another. Therefore, other factors likely help dictate splicing outcomes by regulating these exon circularization events (Kramer, 2015).

Despite key regulatory roles for intronic repeats in multiple eukaryotes, it has been suggested that circular RNA biogenesis in Drosophila melanogaster is not driven by base-pairing interactions (Westholm, 2014). Instead, a positive correlation between the length of the flanking introns and circular RNA abundance was identified in Drosophila (Westholm, 2014). However, the effect of modulating intron lengths on backsplicing has not yet been directly addressed. It is also completely unknown how Drosophila circular RNAs besides Mbl, of which there are >2500 annotated circular RNAs derived from other genomic loci, are generated or post-transcriptionally regulated. Therefore, it is still unclear whether circular RNA biogenesis strategies are conserved across eukaryotes or whether species such as Drosophila use unique mechanisms to determine which exons should be backspliced (Kramer, 2015).

Once produced, circular RNAs are stable transcripts that are naturally resistant to degradation by exonucleases. Two circular RNAs (ciRS7/CDR1as and Sry) modulate the activity of specific microRNAs (Hansen, 2013; Memczak, 2013), but most other RNA circles (in species other than Drosophila) contain few microRNA-binding sites and likely function differently. For example, it has been proposed that many circular RNAs may regulate neuronal functions, and artificial circular RNAs containing an IRES (internal ribosome entry site) can be translated. However, the lack of efficient methods for modulating circular RNA levels or ectopically expressing circular RNAs has limited the ability to define functions for these transcripts (Kramer, 2015).

This study focused on the Drosophila laccase2 gene, as it produces an abundant circular RNA in vitro and in vivo. Evidence is provided that intronic repeats collaborate with trans-acting splicing factors to regulate circularization in flies. Mechanistically, it was found that miniature introns (<150 nt) containing the splice sites and inverted repeats were sufficient to support Laccase2 circular RNA production. The intronic repeats must base-pair to one another for circularization to occur, as has been observed in other eukaryotes. Furthermore, it was found that the strength of these base-pairing interactions dictates whether backsplicing occurs co- or post-transcriptionally: Long flanking repeats appear to allow cotranscriptional processing. Screening a panel of genes, this study found that multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine–arginine) family proteins regulate Laccase2 circular RNA levels in a combinatorial manner. Comparisons with the mbl locus suggest that the circularization mechanisms are distinct, as the Laccase2 circular RNA was not regulated by the Mbl or Laccase2 gene products. Additional circular RNAs were identified that are regulated by unique combinations of hnRNP and SR proteins, suggesting that combinatorial control may be a common regulatory strategy that modulates circular RNA levels. This led to a test of whether this biogenesis mechanism is active in human cells, and it was found that the laccase2 introns can indeed robustly generate circular RNAs. It is thus now possible to efficiently generate "designer" circular RNAs in cells with minimal linear RNA production. In total, the results reveal new insights into how trans-acting factors and intronic repeats collaborate to regulate circular RNA biogenesis across eukaryotes as well as provide new tools for exploring the functions of circular RNAs (Kramer, 2015).

This study demonstrates that intronic repeats and trans-acting hnRNPs and SR proteins combinatorially regulate circularization of the Drosophila laccase2 gene. Base-pairing between transposable elements in the flanking introns facilitates circularization, and the strength of these interactions likely dictates whether backsplicing occurs co- or post-transcriptionally. This mechanism is distinct from the one that regulates Drosophila Mbl circular RNA production (Ashwal-Fluss, 2014) but is similar to that used to generate many circles in humans, mice, and C. elegans. This suggests that base-pairing between intronic repeats may be a major mechanism promoting exon circularization across eukaryotes. Moreover, this study found that the laccase2 exon is dispensable, allowing the laccase2 introns to be used to efficiently generate 'designer' circular RNAs from plasmids in diverse organisms. Altogether, the results suggest that circular RNA biogenesis strategies are conserved across eukaryotes and provide new tools for exploring the functions of circular RNAs (Kramer, 2015).

The current results on the laccase2 locus indicate that base-pairing between complementary intronic sequences efficiently promotes RNA circularization in flies. As the DNAREP1_DM repeats closely flank exon 2 of the laccase2 gene, a model is proposed in which the repeats base-pair to one another, bringing the intervening splice sites into close proximity and facilitating catalysis. The Laccase2 circular RNA then accumulates as one of the most abundant circular RNAs in Drosophila (fifth most abundant across >100 Drosophila RNA sequencing libraries). At the endogenous laccase2 gene locus, the long introns that flank this exon likely slow the overall speed of cotranscriptional splicing, thereby allowing the backsplicing reaction to effectively compete with canonical splicing. Indeed, it was found that the strength of the base-pairing interactions between the flanking introns dictates how quickly backsplicing can occur. When very stable interactions are present, it is possible that exon definition is improved, allowing the rapid and cotranscriptional generation of a circular RNA. Nevertheless, further studies are still required to clarify the exact role that long flanking introns may play in regulating circularization (Kramer, 2015).

Upon examining the introns that flank other abundant Drosophila circular RNAs, this study identified other examples in which complementary regions >60 nt in length flank circularizing exons, including CaMKI, CG11155, CG2052, Parp, and PlexA (which are among the top 25 most abundant Drosophila circular RNAs). Interestingly, the Semaphorin-2b (CG33960) circular RNA (39th most abundant circular RNA) is flanked by introns containing short (CA)_n simple repeats that are complementary to each other over a <30-nt region. Upon cloning a 980-nt region of the Semaphorin-2b pre-mRNA downstream from the pMT, circular RNA production from the plasmid was observed in DL1 cells. Removal of either of the (CA)_n simple repeats, however, strongly reduced circularization. This suggests that diverse inverted repeat sequences, including short simple repeats, may play a general role in facilitating circularization in Drosophila (Kramer, 2015).

Complementary repeats, however, are not observed at all Drosophila loci that generate circular RNAs. Furthermore, many exons that do not circularize are flanked by complementary repeats, so there must be other mechanisms that regulate circularization. This has been most notably demonstrated at the Drosophila mbl locus, which requires the Mbl splicing factor for its circularization. When Mbl protein is in excess, an intricate feedback mechanism is induced: The Mbl protein decreases the production of its own mRNA by binding its pre-mRNA. This blocks canonical splicing and promotes the biogenesis of the Mbl circular RNA, which further functions as a sponge that binds and sequesters the excess Mbl protein. However, this Mbl-driven mechanism appears to be specific for the mbl locus, as this study found that knockdown of the Mbl linear mRNA had no effect on Laccase2, PlexA, or a panel of other circular RNAs. Knockdown of the Laccase2 linear mRNA likewise did not affect Laccase2 circular RNA levels, indicating that the laccase2 locus is not subjected to a similar direct cis-acting feedback mechanism. Instead, it was found that other splicing factors, including hnRNPs and SR proteins, regulate Laccase2 RNA levels (Kramer, 2015).

At the laccase2 locus, it is proposed that hnRNPs (e.g., Hrb27C and Hrb87F) and SR proteins (e.g., SF2 [SRSF1], SRp54 [SRSF11], and B52 [SRSF6]) add an additional layer of control on top of the DNAREP1_DM intronic repeats. Base-pairing between the intronic repeats promotes circularization, but protein binding likely helps ensure that the appropriate ratio of linear to circular Laccase2 RNA is produced. Depletion of any one of these splicing factors alters Laccase2 circle levels, and additive effects were observed when multiple factors were depleted. This suggests combinatorial control, with each protein playing a nonredundant role. Furthermore, Laccase2 circular RNA production does not appear to be linked to exon skipping, and thus these proteins may specifically modulate spliceosome assembly, the speed of splicing, and/or the stability of the mature circular RNA. Notably, it does not seem that Hrb27F, SF2, SRp54, or B52 affects Laccase2 circular RNA stability, as depletion of these factors did not cause the expression of a plasmid-derived Laccase2 circular RNA to increase. It is thus instead proposed that these hnRNPs and SR proteins regulate Laccase2 circular RNA biogenesis (e.g., by binding to the flanking introns or exons), but further studies are required to understand exactly how the intronic repeats and trans-acting factors collaboratively dictate the splicing outcome. Nevertheless, the same SR proteins that regulate the laccase2 locus also regulate the PlexA circular RNA but not the Mbl circular RNA. Since the laccase2 and PlexA exons are both flanked by inverted repeats, it is hypothesized that intronic repeats may generally provide the opportunity for circularization to occur. This is then further regulated by trans-acting factors that combinatorially fine-tune the amount of each circular RNA that the cell ultimately produces (Kramer, 2015).

Catalogs of circular RNAs expressed in various species and cell types have been reported, but the functions for nearly all of these transcripts, including Laccase2, are currently unknown. This is due in part to the current lack of methods for efficiently generating circular RNAs in cells. For example, the circular RNA expression plasmids that have been described all generally produce circular transcripts at a low efficiency (often 20% or less). These plasmids instead generate abundant amounts of linear RNA, which limits their utility for defining circular RNA functions. Using the Drosophila laccase2 and human ZKSCAN1 introns, this study largely overcame this hurdle and generated circular RNAs (ranging in size from 300 to 1500 nt) at a high efficiency in human and fly cells. These transcripts accumulate in the cytoplasm, are resistant to RNase R treatment, and are likely translated when an IRES is present. Furthermore, easy-to-use restriction sites are present in the plasmids, allowing any desired sequence to be queried. Beyond allowing ectopic expression of circular RNAs, these plasmids can be designed to sponge microRNAs or proteins as well as identify novel IRES sequences (Kramer, 2015).

In summary, the current findings provide key insights into how trans-acting factors and intronic repeats regulate circular RNA biogenesis as well as provide new tools for exploring the functions of circular RNAs across eukaryotes. From humans to flies, repetitive elements in introns can act to facilitate backsplicing, but it is still largely unclear why circular RNAs accumulate only in certain tissues. It is hypothesized that base-pairing between repeats is only one part of the "splicing code", and it is ultimately a combination of cis-acting elements and trans-acting splicing factors, including hnRNPs and SR proteins, that dictates whether canonical splicing or backsplicing occurs. Nevertheless, this study has defined a minimal set of elements that is sufficient for promoting efficient exon circularization, which should facilitate the prediction of circular RNAs as well as enable the functions of many circular RNAs to be revealed. Considering that a surprisingly large number of protein-coding genes generates circular RNAs, these previously overlooked transcripts likely represent key ways that gene functions are expanded and modulated (Kramer, 2015).

Protein Interactions

The Plexin family of transmembrane proteins appears to function as repulsive receptors for most if not all Semaphorins. Genetic and biochemical analysis in Drosophila has been used to show that the transmembrane protein Off-track (OTK) associates with Plexin A, the receptor for Sema 1a, and that OTK is a component of the repulsive signaling response to Semaphorin ligands. In vitro, OTK associates with Plexins. In vivo, mutations in the otk gene lead to phenotypes resembling those of loss-of-function mutations of either Sema1a or PlexA. The otk gene displays strong genetic interactions with Sema1a and PlexA, suggesting that OTK and Plexin A function downstream of Sema 1a (Winberg, 2001).

Immunoprecipitated human Plexins A3 and B1 copurify a number of proteins from BOSC-23 cell extracts, some of which became tyrosine phosphorylated in an in vitro kinase assay. Western blotting has indicated that this activity is not due to the presence of Met, Ron, Abl, or Src tyrosine kinases. The most prominent labeled band other than Plexins is approximately of 160 kDa (Winberg, 2001).

To identify the putative Plexin-associated protein, candidates in Drosophila were considered. Several proteins with homology to receptor-tyrosine kinases have been identified that are expressed in the CNS and could potentially interact with Plexins. However, in the cases where the loss-of-function phenotypes have been assayed, there is not a notable similarity with those described for Semaphorins or Plexins, suggesting an unrelated function (e.g., EGFR, FGFR, Derailed). In other cases, in vivo functional data are yet lacking, but some of these candidates may be considered less probable on the basis of molecular weight (e.g., Dror, Nrk). A leading remaining contender is the Drosophila protein Off-track (OTK; previously called Dtrk) (Winberg, 2001).

OTK is a glycoprotein of apparent molecular weight 160 kDa whose extracellular domain, with its six immunoglobulin (Ig) repeats, shows similarity to cell adhesion proteins. In vitro studies have shown that OTK can mediate homophilic adhesion, which results in tyrosine phosphorylation of the intracellular domain (Pulido, 1992). In early Drosophila embryos, OTK transcript is broadly distributed, consistent with both maternal loading and zygotic expression. In later stages, the protein is detected on neuronal cell bodies and axons within the CNS and in the projections of motor neurons as they extend to muscle fibers in the periphery. Because of this axonal localization and in vitro adhesion, OTK has been suggested to play a role in selective fasciculation and axon guidance (Pulido, 1992). Based on its molecular weight, the observation that it can be tyrosine phosphorylated, and its expression on axons at the appropriate time in development to play a role in axon guidance, OTK seemed like a good candidate for possible interaction with Plexin. BLAST searches of protein databases, using either the cytoplasmic kinase or extracellular domain, indicate that the closest relatives of OTK are the chick protein KLG and its human homolog CCK4/PTK7 (Winberg, 2001).

As a first test of OTK protein function, molecular association was examined in COS cells. Epitope-tagged versions of both OTK and a variety of Drosophila and mammalian Plexins (DPlexA, PlexA3, and PlexB1) were generated and tested for expression. The cytoplasmic domains of Plexins are highly conserved, and, thus, binding relationships are likely to be conserved across phylogeny. Cells were cotransfected to express both proteins, and the formation of complexes was analyzed by immunopurification and Western blotting (Winberg, 2001).

Drosophila PlexA (HA tagged) copurifies with immunoprecipitated OTK (myc tagged). Moreover, mammalian PlexA3 and PlexB1 (VSV tagged) also copurify with immunoprecipitated OTK (myc tagged). OTK can copurify all three Plexins but not an unrelated protein, the netrin receptor DCC. In addition, OTK (myc tagged) copurifies with immunoprecipitated mammalian Plexin A3 and B1 (VSV tagged). OTK is copurified in a similar fashion with immunoprecipitated Drosophila Plexin A (HA tagged). These experiments identify OTK as a transmembrane protein that can constitutively associate with both Drosophila and mammalian Plexins in transfected cells, raising the possibility that OTK might play a role in either up- or down-regulating Plexin activity or mediating Semaphorin-Plexin signaling. To determine whether this association reflects a true functional interaction, genetic analysis was performed of OTK in Drosophila (Winberg, 2001).

A direct in vivo test of OTK function was aided by the discovery of a P element insertional mutation near the otk gene, designated EP2017. This mutant strain was obtained from the collection of the Berkeley Drosophila Genome Project and was examined for axon guidance defects in homozygous embryos. Indeed, some defects were found, but they were subtle in nature and poorly penetrant. However, the element is located upstream of the coding sequence and may not completely disrupt gene function. Attempts were made to generate complete loss-of-function otk alleles through imprecise excision of the P element (Winberg, 2001).

The EP2017 element is inserted 30 bp upstream of the 5' end of the published otk cDNA. Since the OTK transcript is ~900 bp longer than the cDNA (Pulido, 1992), it is likely that the insert is in the 5' UTR. Ten excision lines were genetically characterized; eight were homozygous lethal and two homozygous viable (the starting strain is semilethal), suggesting that otk is an essential gene. Molecular analysis indicates that the viable strains otk² and otk⁸ are precise excisions. In contrast, the lethal strain otk³ carries a 3 kb deletion that extends downstream of the EP2017 element, apparently disrupting otk but not upstream genes. The otk³ lesion removes the putative translational start codon and part of the signal peptide and thus likely represents a complete loss-of-function allele. Subsequent examination of axon guidance defects has shown that otk³ and three other lethal alleles are similar to one another in the variety and severity of their phenotypes, which are more pronounced than those displayed by the original EP2017 strain. In comparison, otk² is in the range of wild-type (Winberg, 2001).

These reagents allowed for a test of another property of the EP2017 insert. The EP series of P elements contains a UAS gene-regulatory sequence that, in combination with a GAL4 driver, permits transcription of sequences flanking the insertion site of the P element. In the present case, EP2017 is oriented such that GAL4-regulated expression yields short antisense OTK transcripts. In conjunction with elav-GAL4, one copy of EP2017 produces axon guidance abnormalities comparable with homozygous mutant otk¹ or otk³ strains, suggesting that this antisense transcription from EP2017 confers a neuron-specific dominant loss-of-function phenotype (Winberg, 2001).

If OTK is important for Plexin A function, then loss-of-function mutations in otk might show guidance phenotypes similar to other mutations in the pathway. Specifically, if OTK is a positive activator or effector of Plexin A, then loss-of-function phenotypes of one should resemble loss-of-function phenotypes of the other. However, if OTK is a negative regulator of Plexin A, then the loss of OTK might lead to similar phenotypes as the overproduction of Plexin A protein. Indeed, embryos mutant for otk display axon guidance defects in the CNS and in the projections of the motor nerves, with abnormalities that are similar to those previously reported for PlexA and Sema1a loss-of-function mutants. The projections of motor neurons to their muscle targets are more obviously affected, disrupted in a way that suggests individual growth cones are not always able to defasciculate from pioneer neurons when they should. The most telling examples are provided by the dorsal projections of the segmental nerve (SN) and the ventrolateral or 'b' branch of the intersegmental nerve (ISNb) (Winberg, 2001).

The major projection of the segmental nerve, the SNa, normally extends along the body wall to a lateral position, where it divides into a dorsal and a lateral branch. The dorsal branch then extends further, dividing again and sending fine projections to innervate a group of transverse muscle fibers. In wild-type late stage 16 embryos, the dorsal SNa thus acquires a characteristic 'pitchfork' appearance. In otk loss-of-function or antisense mutants of the same age, these most dorsal growth cones remain fasciculated together in over 60% of segments and extend as a single thicker branch. This is highly similar to the aberrant SNa morphology displayed in Sema1a and PlexA loss-of-function mutant embryos. In contrast, overexpressing Plexin A causes SNa axons to defasciculate prematurely (Winberg, 2001).

The ISNb normally diverges from the main branch of the ISN in a ventral position, termed 'choice point #1'. Within the ventral muscle domain axons of the ISNb then defasciculate from one another: at choice point #2, a single axon splits off to innervate muscle fibers 6 and 7, and at choice point #3, axons either stop and innervate muscle 13 or extend further to muscle 12. By late embryonic stage 16, these growth cones have typically reached their targets and formed rudimentary synaptic contacts along the edges of these muscle fibers. In otk loss-of-function or antisense mutants, growth cones may fail to defasciculate at any of the three choice points. ISNb axons occasionally fail to exit the ISN at choice point #1, instead bypassing their muscle targets completely or else extending small aberrant projections directly from the main branch of the ISN. More often, choice point #1 is navigated correctly but then axons are unable to defasciculate at choice points #2 or #3, resulting in a thickened, stalled nerve and a failure to innervate one or more of the muscles in this domain (Winberg, 2001).

Within the CNS, additional abnormalities are observed. A subset of longitudinal axons is highlighted by monoclonal antibody labeling; in the wild-type, they form neat parallel tracks. In otk mutant embryos, these tracks are variably wavy and defasciculated and occasionally discontinuous. The incidence of 'broken' axon tracks is greater in the antisense embryos than in the loss-of-function embryos (35% versus 15%) (Winberg, 2001).

The abnormalities seen in the SNa and ISNb of embryos lacking otk are qualitatively and quantitatively highly reminiscent of those described for both Sema1a and PlexA mutants. All of these mutants also show qualitatively similar defects in the major axon tracts within the CNS, but, in the case of otk, these defects are less pronounced. Still, the strong resemblance among the phenotypes of all these mutations suggests that these three genes may all be acting in the same genetic pathway, consistent with the hypothesis that OTK positively influences Plexin A function (Winberg, 2001).

Another way to investigate whether these proteins may work together is to test for dominant genetic interactions. For most proteins, reducing gene dose to a single copy (thus reducing the protein level by 50%) produces mild or undetectable defects. However, reducing the gene dose of two different proteins may generate a phenotype if the two proteins normally function together. This 'transheterozygous' genetic test has been applied to several pairs of proteins that have also been shown to interact biochemically: Notch and Delta, Boss and Sevenless, Sema 1a and Plexin A, and Slit and Robo (Winberg, 2001).

Embryos singly and doubly heterozygous for otk and PlexA were examined and strong phenotypic effects due to the combination were observed. Embryos lacking one copy each of both otk and PlexA exhibit the same variety of SNa and ISNb defects as seen in the single homozygous mutants, to nearly the same degree of severity. This provides strong genetic support for the hypothesis that Otk and Plexin A proteins function positively together through direct contact (Winberg, 2001).

Likewise, embryos doubly heterozygous for otk and Sema1a also show phenotypic enhancement beyond additive effects of the single heterozygotes, supporting the idea of a ternary complex of Sema 1a-Plexin A-OTK proteins. However, the severity of phenotypes in the otk, Sema1a combination is somewhat less than in the others. The discrepancy may reflect a true difference between the association of OTK with Sema 1a compared to Plexin A. Alternatively, it may arise from differences in the normal expression levels of the various proteins: if Plexin A were the least abundant component under normal circumstances, then reducing the levels of the other two would be less consequential in this test (Winberg, 2001).

It has been supposed that OTK somehow affects the ability of Plexin A to mediate Sema 1a signaling. However, because all three proteins are expressed by many of the same neurons, the genetic tests above are also consistent with the possibility that OTK may interact directly with Sema 1a in cis. To verify that OTK can act genetically downstream of the signal, use was made of the GAL4 system to misexpress Sema 1a in muscles, thus offering an excess of repulsive target-derived ligand. Ectopic presentation of Sema 1a on specific muscles using UAS-Sema1a and H94-GAL4 turns these muscles into nonpermissive substrates and prevents motoneurons from innervating them correctly. The abnormal innervation of muscle 13 increases from 22% (with H94-GAL4 driver alone) to 49% (with addition of UAS-Sema1a) in this Sema1a gain-of-function experiment. This phenotype is suppressed by removing one copy of otk, reducing neuronal expression levels. Abnormal innervation of muscle 13 is reduced to 26%. It has been shown that the addition of Sema1a increases the percent abnormal from 19% to 53% and removing a single copy of PlexA reduces this frequency of abnormal innervation to 21%. Thus, removal of one copy of otk is nearly as effective in reducing the Sema1a gain-of-function as is removal of one copy of PlexA. Since neuronal OTK is sensitive to muscle-derived Sema 1a, this experiment confirms that OTK is able to act downstream of Sema 1a (Winberg, 2001).

This study has shown that Otk, a transmembrane protein of about 160 kDa, with homology to receptor tyrosine kinases, both associates with Plexins in vitro and appears to function in a Semaphorin-Plexin signaling pathway in vivo to control certain aspects of axon guidance. Biochemical data show that OTK specifically associates with Plexins in vitro. Genetic disruption of otk leads to specific defects resembling those due to lesions in either Sema 1a, a transmembrane Semaphorin that mediates axon defasciculation. These data suggest that all three proteins -- Sema 1a, Plexin A, and OTK -- may function in the same pathway. Genetic interactions suggest that OTK and Plexin A act downstream of Sema 1a. Thus, it appears that OTK and Plexin A can associate as components of a receptor complex that mediates the repulsive signaling in response to Semaphorin ligands (Winberg, 2001).

It is not known whether OTK and Plexins normally associate in vivo in growth cones or whether they might only be brought together by ligand binding. In the absence of ligand in vitro, a tight association is found between the two transmembrane proteins. If transmembrane Semaphorins, like their secreted relatives, function as dimers, then binding of Sema 1a to Plexin A might provide a mechanism for clustering receptor complexes, which by analogy might activate one or more associated kinases and lead to the phosphorylation of Plexin and OTK. Testing such speculations will have to await an appropriate system for testing ligand activation (Winberg, 2001).

Interestingly, despite its homology with receptor tyrosine kinases and the observation that immunoprecipitates of Drosophila OTK possess tyrosine kinase activity (Pulido, 1992), OTK itself is probably not an active tyrosine kinase. The OTK sequence suggests that it belongs to a family of kinase 'dead' receptors. The catalytic domain of OTK, like other members of this family, is altered in a few key conserved residues that are implicated in autophosphorylation (the conserved DFG motif substituted by YPA). Vertebrate family members bear similar alterations in the DFG motif and apparently do not have kinase activity. Modest tyrosine phosphorylation of OTK has been observed in 293T cells but no significant increase in Plexin phosphorylation has been observed upon coexpression with OTK. Thus, OTK either possesses a weak catalytic activity, which is barely detectable in the tested experimental conditions, or like other members of the CCK-4 subfamily of receptor tyrosine kinases, OTK might be kinase dead. In the latter case, some other active kinase would be expected to be present in or recruited to the OTK/Plexin complex in order to account for the observed tyrosine phosphorylation of these proteins. This situation is reminiscent of the interleukin receptors, which are heterodimers composed of a ligand binding subunit and a signal transducing subunit known as gp130. Neither subunit possesses a catalytic activity; rather, gp130 associates with the Janus kinases. Upon ligand binding, the receptors multimerize, resulting in activation of the Janus kinases and tyrosine phosphorylation of the receptor (Winberg, 2001).

Another receptor tyrosine kinase carrying mutations in conserved DFG catalytic residues, h-Ryk/d-Derailed, appears also to be kinase inactive. Nevertheless, Ryk/Derailed is crucially involved in axon guidance. Thus, at least two highly conserved receptor tyrosine kinases, both of which are members of families which are kinase dead -- OTK and Derailed -- have been shown to function in axon guidance. In the case of OTK, it functions apparently by associating with Plexins and helps to mediate their output (Winberg, 2001).

The signal transduction pathway activated by Semaphorins is beginning to be clarified. The cytoplasmic domains of Plexins do not have any obvious signal transduction motif such as a kinase or phosphatase domain. However, the cytoplasmic domains of Plexin B receptors bind directly to the Rac GTPase in a GTP-dependent manner. It has been confirmed that the cytoplasmic domain of Plexin B (PlexB) indeed binds directly to the active, GTP-bound form of the Rac GTPase and, in addition, that a different region of PlexB binds to RhoA. The genetic and biochemical evidence suggests a model whereby PlexB mediates repulsion in part by coordinately regulating two small GTPases in opposite directions: PlexB binds to Rac^GTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA. While the contribution of OTK to this signaling pathway has not yet been investigated, by analogy with other tyrosine-phosphorylated receptor complexes, one hypothesis to test is that a Rho exchange factor is recruited to the activated Plex/OTK complex, providing local activation of Rho (Winberg, 2001).

Prior to the identification of Plexins as Semaphorin receptors and the implication of OTK as a Plexin-associated kinase, both proteins were shown to be capable of mediating cell aggregation in vitro. These studies led to the suggestion that both Plexins and OTK might function as homophilic cell adhesion molecules. Whether either or both of them normally functions in a homophilic fashion in vivo is unknown (Winberg, 2001).

Semaphorins have come to be considered as being ligands and Plexins as their receptors. But their roles in axon guidance may not be this simple. On the one hand, some Semaphorins are transmembrane proteins with cytoplasmic domains that appear as if they might be capable of transducing signals. Thus, some Semaphorins might themselves be receptors as well as ligands. On the other hand, Plexins, which are related to Semaphorins and have extracellular Semaphorin domains, can bind to themselves. Thus, some Plexins might be both ligands and receptors. Finally, Plexins associate with OTK, which also can bind homophilically (Winberg, 2001).

The data presented it this study demonstrate a role for OTK downstream from a Semaphorin on the receiving side of a signaling event. The best evidence for this conclusion is the genetic suppression data. Removing one copy of otk suppresses a Sema 1a gain-of-function phenotype. The most parsimonious interpretation of this result is that OTK functions downstream of Sema 1a. It is not known to what degree OTK binding and function is ligand gated. Moreover, it is not known whether OTK responds directly to Semaphorins, to some other ligand, or alternatively whether it simply binds to Plexins as part of a Semaphorin signaling complex. It will be interesting in the future to determine how these different Semaphorin, Plexin, and OTK proteins associate, modulate Semaphorin-mediated signal transduction, and thus control axon guidance (Winberg, 2001).

Plexin A interacts with MICAL

Members of the semaphorin family of secreted and transmembrane proteins utilize plexins as neuronal receptors to signal repulsive axon guidance. It remains unknown how plexin proteins are directly linked to the regulation of cytoskeletal dynamics. Drosophila MICAL, a large, multidomain, cytosolic protein expressed in axons, interacts with the neuronal plexin A (PlexA) receptor and is required for Semaphorin 1a (Sema-1a)-PlexA-mediated repulsive axon guidance. In addition to containing several domains known to interact with cytoskeletal components, MICAL has a flavoprotein monooxygenase domain, the integrity of which is required for Sema-1a-PlexA repulsive axon guidance. Vertebrate orthologs of Drosophila MICAL are neuronally expressed and also interact with vertebrate plexins, and monooxygenase inhibitors abrogate semaphorin-mediated axonal repulsion. These results suggest a novel role for oxidoreductases in repulsive neuronal guidance (Terman, 2002).

To identify mediators of semaphorin-dependent repulsive axonal guidance, the terminal highly conserved 'C2' portion of the PlexA cytoplasmic domain was used to search for interacting proteins encoded by a Drosophila embryonic (0-24 hr) yeast two-hybrid cDNA library. The strongest interactor has been called MICAL, covers >41 kb of genomic sequence and has at least 25 exons. Based on analysis of isolated cDNAs and Western analysis, there are at least three MICAL isoforms ('long,' 'medium,' and 'short' variants) (Terman, 2002).

Drosophila MICAL is named for its recently characterized vertebrate ortholog, MICAL-1 (for molecule interacting with CasL), which has been shown to associate with CasL and vimentin in nonneuronal cells. Within the plexin-interacting region in the C terminus identified in the screen described in this paper, there is a predicted heptad-repeat, coiled-coil structure. Interestingly, this region of MICAL shares amino acid similarity with several other coiled-coil domain-containing proteins, including a portion of the alpha domain found in the Ezrin, Radixin, and Moesin (ERM) proteins (~22% identity). N-terminal to this domain there is a region rich in prolines, and the last four amino acids of MICAL (ESII) are a PDZ protein binding motif. There are two regions of varying length, with no significant similarity to other proteins, which appear to determine the size of the different MICAL proteins. MICAL has a single LIM domain, a protein-protein interaction module found in a variety of proteins involved in signal transduction cascades and in cytoskeletal organization, and also a single calponin homology (CH) domain, a domain also found in cytoskeletal and signal transduction proteins and known to be involved in actin filament binding. The MICAL N-terminal ~500 aa is highly conserved among MICAL-related proteins but is unique over its entire length in comparison to other proteins (Terman, 2002).

In situ hybridization analysis using RNA probes corresponding to the N or C terminus of MICAL shows that MICAL and PlexA have similar patterns of embryonic mRNA expression. During early Drosophila development (stages 7-8), both are expressed in the ventral neurogenic region and in many nonneuronal tissues (including developing mesoderm, cells surrounding the cephalic furrow and amnioproctodeal invagination, and in gut primordia). This nonneuronal expression is also seen later in embryonic development (stages 11-17), where both are present within the anterior and posterior midgut primordia, the visceral musculature, and weakly in somatic musculature. During axonal pathfinding (stage 13 onward), both are expressed within the developing brain and ventral nerve cord in most, if not all, CNS neurons, but MICAL, like Sema1a and PlexA, is not highly expressed in peripheral sensory neurons (Terman, 2002).

Western blot analysis using a polyclonal antibody directed against the MICAL C terminus (MICAL-CT) has revealed prominent bands at 530 kDa, 330 kDa, 300 kDa, 200 kDa, and 125 kDa in lysates from wild-type embryos that increase in intensity in lysates from embryos harboring a chromosomal duplication that includes the MICAL locus. The three largest protein bands correspond to the predicted molecular weights of the three MICAL cDNAs (Terman, 2002).

MICAL protein is present in neuronal cell bodies, along axons, and in growth cones. MICAL immunostaining first appears in the nervous system at stage 13 and labels motor and CNS projections, and at later embryonic stages, it is present on axons that make up all motor axon pathways: the intersegmental nerve (ISN), the intersegmental nerves b and d (ISNb and ISNd), and the segmental nerves a and c (SNa and SNc). MICAL immunostaining is also present in segment boundaries at the position of muscle attachment sites and at low levels in the lateral cluster of chordotonal organs (Terman, 2002).

MICAL proteins have conserved protein domains with identical organization in all family members and a high degree of amino acid identity among these domains in different MICALs. There is one MICAL in Drosophila and three mammalian MICALs. The MICALs appear unique with respect to containing both calponin homology (CH) and LIM domains, in addition to their conserved N- and C-terminal regions. There is a family of MICAL-like (MICAL-L) proteins, members of which have a similar organization to MICALs but lack the region N-terminal to the CH domain. There is one MICAL-L protein in Drosophila (D-MICAL-L) and at least two family members in humans. D-MICAL-L cDNA and genomic DNA sequence information suggest that D-MICAL-L begins just N-terminal to the CH domain. Analysis of publicly available mammalian cDNA and genomic sequences suggests that human MICAL-L1 and MICAL-L2 are similar in overall domain organization to D-MICAL-L and do not contain the highly conserved ~500 aa MICAL N-terminal domain (Terman, 2002).

The high degree of conservation of the MICAL N terminus among family members (up to 62% identical between flies and humans) suggests that this domain is functionally important. Upon closer examination of the 500 aa conserved N-terminal region, a consensus dinucleotide binding sequence, GXGXXG was found, which is distinct from the sequence present in classical mononucleotide binding motifs. Further, this region contains three separate motifs found in flavoprotein monooxygenases (also called hydroxylases), a subclass of oxidoreductases. The amino acid sequence surrounding the GXGXXG motif matches perfectly the consensus sequence for the ADP binding region of flavin adenine dinucleotide (FAD) binding proteins (Rossmann fold or FAD fingerprint 1), and distinguishes this region from consensus NAD, or NADP binding folds. MICALs also have a well-conserved GD motif (FAD fingerprint 2) C-terminal to the FAD fingerprint 1 region, which is important for binding the ribose moiety of FAD. Finally, MICALs have the conserved DG motif between the FAD fingerprint 1 and 2 motifs that has been reported to be involved in binding the pyrophosphate moiety of FAD. Proteins with these consensus FAD binding regions use FAD in the catalysis of oxidation-reduction reactions. Flavoprotein monooxygenases are oxidoreductases (enzymes that catalyze oxidation and reduction reactions) that catalyze the insertion of one atom of molecular oxygen into their substrate using nucleotides as electron donors. These monooxygenases are also defined by their use of FAD as a coenzyme. Apart from these three consensus regions, monooxygenases vary significantly, reflecting the wide range of enzymes in this family and their variable substrate binding pockets also encompassed within this domain. However, MICALs and other monooxygenases show significant similarity within these three FAD binding regions and also similar spacing of these regions within the monooxygenase domain (Terman, 2002).

Biochemical and genetic analyses strongly suggest that MICALs contain functional FAD binding monooxygenase domains required for mediating plexin signaling. In support of this idea, inhibition of flavoprotein monooxygenase enzymatic activity dramatically attenuates semaphorin-mediated axon repulsion and growth cone collapse. However, though the inhibitor EGCG has a high degree of selectivity for flavoprotein monooxygenases, similar concentrations of EGCG inhibit other enzymes including steroid 5alpha-reductase, NADPH-cytochrome P450 reductase, telomerase, matrix metalloproteinases MMP-2 and MMP-9, and phenol sulfotransferase. Although most of these enzymes are unlikely to be expressed in the growth cones of DRG axons, potential nonspecific effects of these inhibitors cannot be ruled out despite their demonstrated selectivity for monooxygenases. Taken together with the in vivo Drosophila experiments showing a requirement for the MICAL FAD binding region in Sema-1a-mediated axon repulsion, these data suggest redox signaling plays an important role in vertebrate semaphorin-mediated axonal repulsion (Terman, 2002).

Flavoprotein monooxygenases specifically catalyze the oxidation of a number of substrates, and in some contexts they can function as oxidases and generate reactive oxygen species. These results suggest that MICALs are flavoproteins most similar to the flavoprotein monooxygenase family of oxidoreductases, but a complete understanding of the chemical nature of the reactions catalyzed by MICALs awaits future study and identification of substrates. The redox regulation of amino acid residues within signaling proteins (including kinases, phosphatases, small GTPases, guanylate cyclases, and adaptor proteins) and cytoskeletal proteins (including actin, actin binding proteins, intermediate filament proteins, and GAP-43) has been shown to modulate their function. In addition, oxidation of actin leads to disassembly of actin filaments, instability and collapse of actin networks, reduced ability of actin to interact with actin crosslinking proteins, and a decrease in the ability of actin monomers to form polymers. Finally, it is also interesting that MICALs have a putative actin filament binding domain (CH domain) and that MICAL-1 interacts with vimentin, an intermediate filament protein (Terman, 2002).

The proline-rich region of vertebrate MICAL-1 interacts with the SH3 domain of the adaptor protein CasL (HEF1) in nonneuronal cells. CasL, along with the related proteins p130Cas and Efs (Sin), make up the Cas family of proteins that assembles and transduces intracellular signals that stimulate cell migration and axon outgrowth. These proteins have numerous protein-protein interaction domains, including a Src-homology 3 (SH3) domain, multiple SH2 binding sites in their substrate domain, several proline-rich motifs, and a C-terminal dimerization module. This structure suggests a role for Cas family proteins as docking molecules, and numerous interacting proteins have been identified, including kinases (e.g., FAK, Src, and Abl), phosphatases (e.g., PTP-1B and SHP2), GEFs (e.g., C3G), and adaptor proteins (e.g., Nck, Crk, Grb2, and 14-3-3). Studies indicate that Cas proteins localize mainly to focal adhesions and stress fibers and that they are required in integrin-dependent cell migration and actin filament assembly. Cas proteins, therefore, may play an important role in plexin-mediated repulsive and attractive guidance events (Terman, 2002).

Plexin and stereotyped pruning of long hippocampal axon branches

Like naturally occurring neuronal cell death, stereotyped pruning of long axon branches to temporary targets is a widespread regressive phenomenon in the developing mammalian brain that helps sculpt the pattern of neuronal connections. The mechanisms controlling stereotyped pruning are, however, poorly understood. Evidence that semaphorins, activating the Plexin-A3 receptor, function as retraction inducers to trigger-stereotyped pruning of specific hippocampal mossy fiber and pyramidal axon branches. Both pruning events are defective in Plexin-A3 mutants, reflecting a cell-autonomous requirement for Plexin-A3. The distribution of mRNAs for Sema3F and Sema3A makes them candidates for triggering the pruning. In vitro, hippocampal neurons respond to semaphorins by retracting axon branches. These results implicate semaphorins as retraction inducers controlling stereotyped pruning in the mammalian brain (Bagri, 2003).

These studies support the existence of retraction inducers as triggers for stereotyped pruning in vivo, and identify class 3 semaphorins, functioning via neuropilin/plexin receptor complexes, as mediators of this function. In vivo analysis provides strong evidence for control of stereotyped pruning of two temporary hippocampal projections by a plexin-dependent mechanism. The stereotyped pruning of the projection from CA1 pyramidal neurons in the hippocampus to the medial septum, which normally occurs by P5, is significantly impaired in P8 Plexin-A3 mutant mice, as assessed by retrograde labeling. Because this labeling method does not allow for quantitative assessment, it is difficult to determine the extent of the defect. However, qualitative comparison of the extent of labeling before and after the normal pruning period (P0 and P8, respectively) suggests that a large fraction of the projection -- if not the entire projection -- fails to prune (Bagri, 2003).

The extent of defective pruning can be more easily assessed in the case of the infrapyramidal bundle, which can be directly visualized. This bundle is initially long, extending about two-thirds the length of CA3, then shortens dramatically between P20 and P30 to assume its adult length. This occurs without obvious changes in dentate granule cell neurogenesis or apoptosis, consistent with shortening occurring by pruning. Interestingly, IPB pruning occurs seemingly stochastically between P20 and P30, suggesting that multiple mechanisms regulate the pruning -- perhaps even activity-dependent mechanisms. In Plexin-A3 mutants, however, no evidence of pruning was observed during the normal pruning period, or even as late as P60. Thus, Plexin-A3 is absolutely required for infrapyramidal bundle pruning to occur (Bagri, 2003).

Since Plexin-A3 is expressed in many different cells of the hippocampus during its development, the pruning defects observed in Plexin-A3 mutant mice could reflect a cell-autonomous role for Plexin-A3 or a non-cell-autonomous role. X-linked mosaic analysis, however, supports a cell-autonomous role for Plexin-A3-and thus a receptor role for Plexin-A3- in both IPB and hippocampal-septal pruning (Bagri, 2003).

Together, the results support the idea that Plexin-A3 functions cell autonomously to regulate the pruning process directly. Furthermore, the results suggest that Plexin-A3 functions as a component of a receptor complex to transduce a semaphorin-induced pruning signal. In the case of the infrapyramidal bundle, the evidence comes from the observation that a similar pruning defect is observed in mutants for Neuropilin-2, which encodes a receptor that complexes with Plexin-A3 to transduce the Sema3F signal, and the finding that Sema3F is expressed in the region where the IPB prunes back. Together, these observations strongly support a role for Sema3F in stimulating pruning of the IPB by activating a Neuropilin-2/Plexin-A3 receptor complex. This prediction is borne out by a recent study of the Sema3F knockout mouse and, as predicted, an overextension of the infrapyramidal bundle was found in the adult mutants, similar to that observed in Neuropilin-2 and Plexin-A3 mutants (Bagri, 2003).

The cell biological mechanisms through which stereotyped axonal pruning occurs in vivo remain uncharacterized. The possibility has been raised that pruning occurs in vivo through regulated axonal degradation or degeneration, as has been seen in vitro in response to neurotrophin withdrawal or treatment with ephrins. However, it is also possible that the pruning occurs by axonal retraction. Indeed, retraction rather than degeneration appears to mediate the pruning of hippocampo-septal axons in vitro: in both explant cultures in collagen gels and dissociated cell cultures, the axons of CA1 neurons treated with Sema3A appear to retract, and analysis at intermediate time points fails to reveal any blebbing of axons that is characteristically associated with degeneration. It should be emphasized that these in vitro observations do not prove that pruning in vivo is caused by retraction rather than degeneration. However, failure to detect silver-stained product during infrapyramidal bundle pruning in vivo argues against degeneration being the mode of IPB axonal pruning. Alternatively, if degeneration occurs, it must be sufficiently different from pathological degeneration to prevent the formation of a silver stain reaction product (Bagri, 2003).

Nervy links protein kinase a to plexin-mediated semaphorin repulsion

Cyclic nucleotides regulate axonal responses to a number of guidance cues through unknown molecular events. Drosophila nervy, a member of the myeloid translocation gene family of A kinase anchoring proteins (AKAPs), regulates repulsive axon guidance by linking the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) to the Semaphorin 1a (Sema-1a) receptor Plexin A (PlexA). Nervy and PKA antagonize Sema-1a-PlexA-mediated repulsion, and the AKAP binding region of Nervy is critical for this effect. Thus, Nervy couples cAMP-PKA signaling to PlexA to regulate Sema-1a-mediated axonal repulsion, revealing a simple molecular mechanism that allows growing axons to integrate inputs from multiple guidance cues (Terman, 2004). Subsequent analysis has shown that Nervy is a member of the MTG protein family and probably functions in the nucleus as transcriptional corepressor. Although a cytoplasmic function for Nervy, described in this section of The Interactive Fly, cannot be ruled out, it is suggested that the axonal migration phenotypes observed in nervy mutant Drosophila embryos may be due to alterations in gene expression rather than a failure to anchor PKA to the plasma membrane (Ice, 2005). Nervy, like PlexA, is highly expressed in the Drosophila embryonic central nervous system (CNS), including in motor neurons (Feinstein, 1995) and their axons. An antibody to a conserved region of mammalian MTG proteins also identified Drosophila Nervy within CNS and motor axons. Immunoprecipitation of hemagglutinin (HA) epitope–tagged neuronal PlexA from Drosophila embryonic lysates revealed associated Nervy, and neuronal HA-PlexA was detected in immunoprecipitates of Nervy, which suggests that nervy and PlexA interact in neurons. Nervy also immunoprecipitates with PKA RII in Drosophila embryos, and an epitope (Myc) tagged neuronal nervy immunoprecipitated with Drosophila PKA RII, which indicates that nervy is a neuronal AKAP (Terman, 2004).

If Nervy serves to tether PKA to the PlexA receptor, then type II PKA should associate in a complex with PlexA. An antibody specific for PKA RII decorates embryonic Drosophila CNS and motor axons, and PKA RII coimmunoprecipitated (co-IP) with HA-PlexA expresses in neurons, showing that type II PKA is associated with the PlexA receptor complex. pka RII LOF mutant embryos also exhibit highly penetrant axon guidance defects that closely resemble the guidance defects observed in nervy LOF, PlexA GOF, and MICAL GOF mutants. In addition, pka RII LOF mutants, like nervy LOF mutants, enhance the repulsive effects of Sema-1a, which suggests that type II PKA antagonizes Sema-1a repulsive axon guidance (Terman, 2004).

To test the necessity of nervy-type II PKA interactions in regulating Sema-1a-PlexA signaling, a single amino acid substitution of a proline for a valine residue was made in Nervy (nervy^V523P) that was analogous to a mutation that disrupts MTG16-PKA RII interactions. Transgenic flies were generated expressing epitope (myc)-tagged nervy^V523P, but unlike neuronal expression of wild-type nervy in a nervy LOF mutant background, neuronal nervy^V523P failed to rescue the nervy LOF mutant phenotypes. Therefore, it was reasoned that nervy^V523P might function in a dominant-negative manner by retaining its ability to bind to PlexA but blocking the coupling of PKA to PlexA. Indeed, expression of myc-nervy^V523P in all neurons in a wild-type background results in axon guidance phenotypes similar to those seen in nervy or pka RII LOF mutants. These phenotypes are the opposite of those seen when wild-type Nervy is expressed in all neurons and are indicative of increased Sema-1a-PlexA repulsion because they resemble MICAL and PlexA GOF mutants. These results suggest that nervy's ability to bind type II PKA is critical for the modulation of Sema-1a-PlexA repulsive guidance (Terman, 2004).

14-3-3ε couples protein kinase A to semaphorin signaling and silences plexin RasGAP-mediated axonal repulsion

The biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are among the largest families of axon guidance cues and utilize Plexin (Plex) receptors to exert repulsive effects on axon extension. However, Semaphorin repulsion can be silenced by other distinct cues and signaling cascades, raising questions of the logic underlying these events. This study uncovers a simple biochemical switch that controls Semaphorin/Plexin repulsive guidance. Plexins are Ras/Rap family GTPase activating proteins (GAPs) and this study finds that the PlexA GAP domain is phosphorylated by the cAMP-dependent protein kinase (PKA). This PlexA phosphorylation generates a specific binding site for 14-3-3ε, a phospho-binding protein that is necessary for axon guidance. These PKA-mediated Plexin-14-3-3ε interactions prevent PlexA from interacting with its Ras family GTPase substrate and antagonize Semaphorin repulsion. These results indicate that these interactions switch repulsion to adhesion and identify a point of convergence for multiple guidance molecules (Yang, 2012).

Axons rely on the activation of guidance receptors for correct navigation but receptor inactivation is also thought to be a means through which growth cones integrate both attractive and repulsive guidance signals. The current results indicate that such a mechanism plays a critical role in Sema/Plex-mediated repulsive axon guidance. PlexA was found to use its GAP activity to specify axon guidance but this activity is antagonized by a PKA-mediated signaling pathway. PKA directly phosphorylates the GAP domain of PlexA and this phosphorylation provides a binding site for 14-3-3ε. 14-3-3ε is critical for axon guidance and disrupts the ability of PlexA to interact with its Ras GTPase substrate. These interactions effectively switch PlexA-mediated axonal repulsion to Integrin-mediated adhesion and provide a simple biochemical mechanism to integrate antagonistic axon guidance signals (Yang, 2012).

Genetic experiments identify a critical role for 14-3-3ε proteins in directing axon guidance events during development. The 14-3-3 proteins are a phylogentically well-conserved family of cytosolic signaling proteins including seven mammalian members that play key roles in a number of cellular processes. Interestingly, 14-3-3 family proteins were first identified because of their high level of expression in the brain, but despite considerable interest in their functions, their roles in the nervous system are still incompletely understood. For instance, 14-3-3 proteins are highly expressed in growing axons and have been found to modulate neurite extension and growth cone turning in vitro in a number of contexts. However, their necessity for directing axonal growth and guidance events in vivo are unknown as is the functional role of each family member in these neurodevelopmental processes. This study found that one of the two Drosophila 14-3-3 family members, 14-3-3ε, is required in vivo for axon guidance and plays specific roles in the pathfinding of motor and CNS axons. Moreover, previous mutant analysis has revealed that the other 14-3-3 family member in Drosophila, 14-3-3ζ (Leonardo), does not exhibit significant motor axon guidance or innervation defects but plays a critical role in synaptic transmission and learning and memory. These results indicate that individual 14-3-3 family members play specific roles in the development of the nervous system and in light of the requirement of 14-3-3ε in mammalian brain development and neuronal migration, and potential roles for 14-3-3ε (YWHAE) in human neurological disease, future work will determine if 14-3-3ε's role in axon guidance is phylogenetically conserved (Yang, 2012).

Genetic and biochemical experiments also identify a specific role for 14-3-3ε in regulating Sema/Plex-mediated repulsive axon guidance. Sema/Plex-mediated repulsive axon guidance is antagonized by increasing cAMP levels, but the mechanisms underlying these cAMP-mediated effects are poorly understood. Interestingly, Plexins associate with the cAMP-dependent protein kinase (PKA) via MTG/Nervy family PKA (A kinase) anchoring proteins (AKAPs). AKAPs position PKA at defined locations to allow for the spatially and temporally specific phosphorylation of target proteins in response to local increases in cAMP and this study now finds that PKA phosphorylates the cytoplasmic portion of PlexA. Genetic and biochemical results suggest that this phosphorylation provides a binding site for a specific 14-3-3 family member, 14-3-3ε. 14-3-3 proteins are well known as phosphoserine/threonine-binding proteins and have been found to utilize this ability to regulate the activity of specific enzymes. This study found that mutating the 14-3-3ε binding site on PlexA generates a hyperactive PlexA receptor, providing a better understanding of the molecular and biochemical events through which cAMP signaling regulates Sema/Plex repulsive axon guidance. Future work will focus on identifying the upstream extracellular signal that increases cAMP levels, although it is interesting that the axonal attractant Netrin is known to increase cAMP levels and antagonize Sema-mediated axonal repulsion (Yang, 2012).

The results also indicate that the GAP activity of PlexA is critical in vivo for repulsive axon guidance and that cAMP/PKA/14-3-3ε signaling regulates this Plexin RasGAP-mediated repulsion. Plexins are GAPs for Ras family proteins and in vitro work has revealed that the GAP activity of Plexin is important for its signaling role. This study now finds that RasGAP activity is required in vivo in neurons for Plex-mediated repulsive axon guidance. Moreover, the results indicate that 14-3-3ε binds to a single phosphoserine residue within the PlexA GAP domain and antagonizes PlexA RasGAP-mediated axon guidance. Interestingly, positive regulation of GTPase signaling may be a conserved function for 14-3-3ε since it also increases the efficiency of Ras signaling during Drosophila eye development and 14-3-3 turns off the activity of other known GAPs and enhances Ras signaling. Therefore, the results suggest a model in which Sema/Plex interactions activate PlexA GAP activity, which inactivates Ras/Rap and disables Integrin-mediated adhesion. However, these Sema/Plex-mediated effects are subject to regulation, such that increasing cAMP levels activates PlexA-bound PKA to phosphorylate PlexA and provide a binding site for 14-3-3ε. These PlexA-14-3-3ε interactions occlude PlexA GAP-mediated inactivation of Ras family GTPases and restore Integrin-dependent adhesion (Yang, 2012).

In conclusion, this study has identified a simple mechanism that allows multiple axon guidance signals to be incorporated during axon guidance. Neuronal growth cones encounter both attractive and repulsive guidance cues but the molecular pathways and biochemical mechanisms that integrate these antagonistic cues and enable a discrete steering event are incompletely understood. One way in which to integrate these disparate signals is to allow different axon guidance receptors to directly modulate each other's function. Another means is to tightly regulate the cell surface expression of specific receptors and thereby actively prevent axons from seeing certain guidance cues. Still further results are not simply explained by relatively slow modulatory mechanisms like receptor trafficking, endocytosis, and local protein synthesis but indicate that interpreting a particular guidance cue is susceptible to rapid intracellular modulation by other, distinct, signaling pathways. The results now indicate a means to allow for such intracellular signaling crosstalk events and present a logic by which axon guidance signaling pathways override one another. Given this molecular link between such key regulators of axon pathfinding as cyclic nucleotides, phosphorylation, and GTPases, the observations on silencing Sema/Plex-mediated repulsive axon guidance also suggest approaches to neutralize axonal growth inhibition and encourage axon regeneration (Yang, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

Embryonic expression of PlexA and PlexB was examined by Northern analysis and by mRNA in situ hybridization. PlexA probes detect a single transcript of about 7.5 kb; PlexB probes detect two transcripts, approximately 8 and 12 kb. PlexA and PlexB mRNAs show largely similar in situ localization: both are maternally deposited and are broadly distributed during early embryogenesis. Beginning with germband retraction, both transcripts show a reduction in general expression, while remaining highly expressed in the CNS. After embryonic stage 15, during the period in which many motor axons are reaching their targets, PlexA and PlexB transcripts are largely confined to the CNS, where they are expressed by many neurons. Although transcripts are found in myoblasts, expression does not persist in muscle fibers (Winberg, 1998).

Larval

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA⁺ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

In contrast to the histolyzing larval muscles, the CNS undergoes dramatic differentiation and restructuring during early metamorphosis. The majority of the CNS is composed of adult-specific neurons that reorganize at this time by extending processes and establishing new connections. Several genes known to be involved in neuronal-specific processes are differentially regulated during the late larval ecdysone pulse (see Developmental control genes induced during metamorphosis.) For example, the Drosophila neurotactin and plexin A genes are induced. These genes are involved in axonal pathfinding and in establishing synaptic connections. The neurotactin (nrt) gene product is involved in growth cone guidance and is localized to the cell surface at points of interneuronal cell contact in the presumptive imaginal neurons within the larval CNS. Nerve cord condensation does not occur normally in the late third instar CNS of nrt mutant animals. In prepupae, nrt is expressed in a tissue- and cell type-specific manner: it is restricted to a small set of ocellar pioneer neurons in the brain, photoreceptors of the eye, and some sensory neurons in the developing wing. It is suggested that nrt, like the control genes induced from >18 hours BPF to PF, is regulated by the late larval ecdysone pulse. The plexin A gene belongs to a family of genes that encode Ca²⁺-dependent homophilic cell adhesion molecules first identified in the vertebrate CNS and PNS. Drosophila Plexin A also acts as a receptor for class I semaphorins, and both loss of function and overexpression experiments demonstrate that Plexin A is involved in axon guidance and repulsion of adjacent neurons (defasciculation). Many neurons defasciculate in response to ecdysone during nervous system remodeling, and it is suggested that an increase in plexin A expression may be partly responsible for this response. Several more differentially expressed neuronal-specific molecules are shown at The Drosophila Microarray Project. These genes provide several new candidates for factors that are involved in the neuronal outgrowth and morphological remodeling responses to ecdysone (White, 1999).

Multiple interactions control synaptic layer specificity in the Drosophila visual system

How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. This study demonstrates that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, these results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers (Pecot, 2013).

Although the growth cones of L1, L3, and L5 neurons target to different layers, they initially overlap within a common domain in the outer medulla. Based on biochemical interactions and the mistargeting phenotypes and protein expression patterns described in this paper, it is envisioned that CadN-dependent adhesive interactions restrict processes to the outer medulla and that PlexA-expressing tangential neurons prevent Sema-1a expressing growth cones from projecting into the inner medulla. L2 and L4 growth cones also appear to initially target to a common domain within the distal outer medulla, but do not require Sema-1a and CadN for this targeting step and thus utilize an alternative mechanism. Interestingly, the morphology of L2 and L4 neurons does rely on Sema-1a and CadN function, indicating that within lamina neurons, these molecules regulate different aspects of targeting. This is supported by the expression of Sema-1a and CadN in all lamina neuron subclasses during development (Pecot, 2013).

In mice separate channels encoding light increments (ON) and decrements (OFF) are spawned in the outer retina and relayed to different sublaminas of the inner plexiform layer (IPL). The current findings are reminiscent of recent studies in the mouse IPL (Matsuoka, 2011) in which Kolodkin and colleagues demonstrated that the processes of different subclasses of PlexA4-expressing amacrine cells are segregated to different OFF layers and that this requires both PlexA4 and Sema6A. Although these proteins act in a more traditional fashion as a receptor and ligand, respectively, they are expressed in a complementary fashion early in development when the developing neuropil is very thin, with PlexA expressed in the nascent OFF layer and Sema6A in the developing ON layers. This raises the intriguing possibility that, as in the medulla, different cells initially target to common domains, from which they then segregate into discrete layers. As Cadherin proteins are differentially expressed in a layered fashion in the developing IPL and defects in targeting are incomplete in both Sema6A and PlexA4 mutants (Matsuoka, 2011), it is possible that, as in the medulla, Semaphorin/Plexin repulsion acts in parallel with cadherin-based adhesion to control layer-specific patterning within the developing IPL (Pecot, 2013).

Taken together, these studies suggest that the restriction of processes to a common domain prior to their segregation into distinct layers may be a developmental strategy used in both the medulla and the vertebrate IPL. This step-wise process may represent a more general strategy for reducing the molecular diversity required to establish synaptic connections by limiting the potential synaptic partners that growth cones and nascent dendritic arbors encounter within the developing neuropil (Pecot, 2013).

After targeting to a common domain within the outer medulla, L3 growth cones undergo stereotyped changes in shape and position that lead to segregation into the M3 layer. Initially, L3 growth cones are spear-like, spanning much of the depth of the incipient outer medulla. They then expand and elaborate a myriad of filopodia before resolving into flattened synaptic terminals within the M3 layer. This transformation is marked by two prominent steps: extension of processes from one side of the lateral region of the growth cone into the incipient M3 layer and retraction of the leading edge of the growth cone from the incipient M5 layer (part of the domain shared by L1 and L5 growth cones) (Pecot, 2013).

It has been suggested that CadN may regulate the extension within M3, as this step is partially perturbed in CadN mutant growth cones. However, as CadN mutations affect the initial position of L3 growth cones within the outer medulla, the extension defect within the M3 layer may be indirect. By contrast, in sema-1a mutant growth cones, initial targeting is indistinguishable from wild-type, so defects in retraction away from the incipient M5 layer are likely to reflect a direct role for Sema-1a in this later step in growth cone reorganization. PlexA RNAi phenocopies a sema-1a null mutation and, thus, PlexA is also required for retraction and is likely to function on medulla tangential fibers, where it is most strongly expressed. In support of this, the tip of the L3 growth cone that retracts is in close proximity to these PlexA-expressing fibers (Pecot, 2013).

The function of Sema-1a/PlexA signaling in sculpting L3 growth cones appears to be distinct mechanistically from the earlier role it plays in confining the growth cones to a common domain. During initial targeting, PlexA acts as a barrier to L3 growth cones and prevents them from projecting beyond the outer medulla. Thus, at this early step, Sema-1a/PlexA interaction provides a stop signal for the leading edge of L3 (uncovered in double mutants with CadN). In the second step, however, Sema-1a/PlexA signaling promotes retraction into the M3 layer. How these diverse outputs of Sema-1a/PlexA signaling arise is unclear. Sema-1a may be coupled to different downstream effectors at each step, modified by association with other receptor subunits, or may be modulated by other extracellular signaling pathways (Pecot, 2013).

CadN may also play a role in the retraction of L3 growth cones away from the domain shared with L1 and L5 growth cones. In early pupal stages, disrupting CadN function, while leaving growth cone morphology largely spear-like, causes L3 axons to project deeper within the medulla. Under these conditions, Sema-1a function is sufficient to prevent the growth cones from extending beyond the outer medulla. Subsequently, CadN mutant L3 growth cones fail to move away from the outer medulla's proximal edge into the developing M3 layer and thus remain within the most proximal layer, M6. This suggests that CadN, while acting in parallel with Sema-1a to restrict L3 growth cones to the outer medulla initially, may also be required at later stages for movement of the L3 leading edge into the M3 layer. As CadN has been shown previously to regulate neurite outgrowth over cultured astrocytes, it may be required for L3 growth cones to move along adjacent processes. However, the initial projection of L3 axons into the medulla is not affected by CadN mutations, indicating that other components control this process. It also remains possible that the defect in growth cone retraction results indirectly from CadN's earlier role in targeting; this earlier role may account for the defects in growth cone extension within M3 (Pecot, 2013).

Disrupting CadN function in different neurons affects targeting in unique ways. For example, L5 axons lacking CadN target to the proper layer, but extend inappropriately within the layer into neighboring columns (Nern, 2008). In addition, CadN mutant R7 growth cones display abnormal morphology and, in contrast to mutant L3 growth cones, initially target correctly, but retract to a more superficial medulla region. Collectively, these findings demonstrate that CadN regulates divergent features of growth cone targeting in different contexts. This likely reflects molecular diversity between different growth cones and illustrates the importance of understanding how molecules act in combination to generate target specificity (Pecot, 2013).

These studies add to previous findings suggesting that column assembly relies on a precisely orchestrated sequence of interactions between different neuronal cell types (Nern, 2008; Timofeev, 2012). This study shows that, as L1, L3, and L5 growth cones expressing Sema-1a enter the medulla, they meet the processes of newly arriving tangential fibers expressing PlexA, which acting in parallel with CadN, prevents extension of these growth cones into the inner medulla. This timing may permit other Sema-1a-expressing growth cones to extend into the inner medulla at earlier stages; these growth cones may then use Sema-1a/PlexA signaling for patterning connections in the inner medulla or deeper neuropils of the lobula complex. Subsequent sculpting of the L3 growth cone, mediated by Sema-1a/PlexA and perhaps CadN, leads to its reorganization into an expanded terminal within M3. As L3 growth cones become restricted to the M3 layer, Netrin, secreted from L3 growth cones, becomes concentrated within the M3 layer, and this, in turn, attracts R8 growth cones to the M3 layer, as recently described by Salecker and colleagues (Timofeev, 2012; Pecot, 2013 and references therein).

Given the extraordinary cellular complexity of the medulla neuropil, with over 100 different neurons forming connections in different medulla layers, and the few mechanistic clues to layer specific targeting that have emerged so far, a complex interplay between different sets of neurons is envisioned to be required to assemble the medulla circuit. The availability of specific markers for many of these neurons, techniques to follow the expression of even widely expressed proteins at the single cell level as is described in this study, and the ability to genetically manipulate single cells during development provide a robust system for uncovering the molecular logic regulating the layered assembly of axon terminals, dendritic arbors, and synaptic connectivity (Pecot, 2013).

EFFECTS OF MUTATION

If Plexins are indeed Semaphorin receptors, then lesions in the PlexA gene might be expected to show similar axon guidance defects as are displayed by embryos mutant for either Sema1a (Yu, 1998 ) or Sema2a (Winberg, 1998). In embryos lacking PlexA, axon guidance defects were found both in the CNS and in the projections of motor nerves to their muscle targets in the periphery. These embryos do not show any morphological abnormalities, muscle defects, or cell fate changes. Embryos from the Df(4)G strain (that does not remove PlexA) display normal axon guidance, indicating that the observed phenotypes are closely linked with the PlexA gene. To test whether axon guidance phenotypes associated with the deficiency are actually due to the lack of PlexA, expression was restored using a transgenic construct, UAS-PlexA, under the transcriptional control of the neuron-specific driver, elav-GAL4. This combination rescues motor and CNS axon guidance defects in homozygous deficiency embryos. Some segments are not completely restored to wild type, but rather than displaying loss-of-function phenotypes, these segments display gain-of-function phenotypes for PlexA. Thus, it is concluded that the aberrations seen in the deficiency strain do indeed result from the lack of PlexA. Moreover, neuron-specific replacement of PlexA is sufficient to rescue the observed guidance phenotypes (Winberg, 1998).

PlexA-deficient embryos show axon guidance phenotypes that markedly resemble defects seen in Sema1a loss-of-function mutants (Yu, 1998). Such defects are seen in the 'b' branch of the intersegmental nerve (the ISNb), which innervates ventral muscles. ISNb axons normally exit the CNS as part of the ISN; they defasciculate from and exit the ISN at ISNb choice point 1, entering the ventral muscle region as a fasciculated bundle. At ISNb choice point 2, a single axon leaves the ISNb to innervate muscle fibers 6 and 7. At ISNb choice point 3, certain growth cones extend further dorsally to innervate muscle 12, while others stop and innervate muscle 13. In the absence of PlexA, ISNb growth cones often fail to defasciculate from one another at any or all three of the ISNb choice points. Occasionally they fail to exit the ISN and thus bypass the ventral muscles; in some cases they innervate their ventral muscle targets via small projections made directly from the main branch of the ISN. More often, they exit the ISN but then fail to defasciculate from each other at choice points 2 and/or 3, leading to a thickened, stalled nerve branch and failure to innervate muscles 6 and 7 and/or 12 (Winberg, 1998).

In addition to the ISNb, the segmental nerve (SN) is also frequently abnormal, with defects resembling those of Sema1a mutants (Yu, 1998 ). In wild-type embryos, the SN exits the CNS, and its main branch, the SNa, extends past the ventral muscle domain to the lateral muscle region. The SNa then divides into a lateral and a dorsal branch at SNa choice point 1, and then further dorsally, bifurcates again at SNa choice point 2. In PlexA-deficient embryos examined at late stage 16, axons at the second choice point failed to defasciculate from one another in roughly 70% of segments and instead extended dorsally as a single branch (Winberg, 1998).

Projections within the CNS are also abnormal in both Sema1a and PlexA mutants. Three major longitudinal axon fascicles on each side of the CNS can be detected with the 1D4 monoclonal antibody against Fas II. In wild-type embryos these tracts are evenly spaced and show fairly uniform thickness. In embryos lacking PlexA, the outermost longitudinal Fas II-positive fascicle is often disrupted, being thinner in some segments, discontinuous in others, and sometimes fused with the middle Fas II-positive fascicle (Winberg, 1998).

This analysis of phenotypes in PlexA Df mutant embryos (and their rescue by a PlexA transgene) clearly indicates an important role for PlexA in axon guidance. The phenotypes for PlexA in the projections of the ISNb, the SNa, and within the CNS are strikingly similar, both qualitatively and quantitatively, to those reported for null mutations of Sema1a (Yu, 1998 ). The high degree of phenotypic correspondence strongly suggests that these two genes encode components of the same pathway. Because Sema 1a has been described as a repulsive ligand for growth cone guidance, it is proposed that PlexA functions as a Sema 1a receptor for these guidance events (Winberg, 1998).

A further loss-of-function phenotype for PlexA is seen in the projection of the transverse nerve (TN). This nerve is composed of two parts, a peripheral neuron that extends an axon toward the CNS, and a pair of central neurons that project outward. These two projections normally extend toward one another along a shared mesodermal substrate, meeting and fasciculating near muscle 7. In PlexA-deficient embryos, growth cones from the TN extend ectopic projections onto ventral muscles in 36.5% of segments, compared with 2.8% in the PlexA rescue background. This phenotype is not seen in Sema1a mutants, raising the possibility that PlexA may also interact with one or more additional ligands (Winberg, 1998).

One way to check the hypothesis that PlexA functions as a Sema 1a receptor is to test for dominant genetic interactions between the two genes. In most cases, reducing gene dosage by one copy (thus reducing protein by 50%) has little phenotypic effect. However, simultaneously reducing the dose of two genes whose protein products function together may sufficiently impair their combined function such that phenotypes appear. Such a ''transheterozygous'' phenotype has been demonstrated for various ligand-receptor pairs in Drosophila. Embryos heterozygous for either or both Sema1a and PlexA were examined and significant enhancement was found in embryos in which both were heterozygous. Each of the phenotypes described above for the ISNb, SNa, and CNS is recapitulated in the double heterozygotes. For example, removing one copy of either Sema1a or PlexA permits nearly wild-type levels of ventral muscle innervation by ISNb neurons. Removing both copies of either gene leads to abnormal innervation in most segments. Removing one copy each of Sema1a and PlexA causes the same repertoire of defects in a similar proportion of segments as the single homozygous mutants. Likewise, the rate of defasciculation failures in the dorsal branch of the SNa is almost the same in the transheterozygous combination as it is in the Sema1a or PlexA homozygous mutants alone, roughly 70%. The fraction of affected segments within the CNS is smaller in the transheterozygotes (20% compared to 50% in Sema1a or PlexA) but still much more than would be expected from simple addition (<10%). These results strongly suggest that Sema1a and PlexA are in the same pathway and further suggest a direct physical interaction between the two proteins (Winberg, 1998).

The Drosophila receptor Guanylyl cyclase Gyc76C is required for Semaphorin-1a-Plexin A-mediated axonal repulsion

Cyclic nucleotide levels within extending growth cones influence how navigating axons respond to guidance cues. Pharmacological alteration of cAMP or cGMP signaling in vitro dramatically modulates how growth cones respond to attractants and repellents, although how these second messengers function in the context of guidance cue signaling cascades in vivo is poorly understood. Using a novel Sema-1a-dependent forward genetic screening approach, it was found that Drosophila receptor-type guanylyl cyclase: Gyc76C, a protein possessing a single transmembrane domain, is required for semaphorin-1a (Sema-1a)-plexin A repulsive axon guidance of motor axons in vivo. Genetic analyses define a neuronal requirement for Gyc76C in axonal repulsion. Additionally, it was found that the integrity of the Gyc76C catalytic cyclase domain is critical for Gyc76C function in Sema-1a axon repulsion. These results support a model in which cGMP production by Gyc76C facilitates Sema-1a-plexin A-mediated defasciculation of motor axons, allowing for the generation of neuromuscular connectivity in the developing Drosophila embryo (Ayoob, 2004).

These experiments provide an important molecular link between semaphorin-mediated repulsion and cGMP signaling in vivo. Gyc76C is critical for Sema-1a-Plexin A-mediated selective defasciculation of axon bundles in the developing Drosophila neuromuscular system. A conserved amino acid residue within the Gyc76C cyclase domain, a residue required for receptor guanylyl cyclase (rGC) catalytic activity, is also required in Gyc76C for correct motor axon pathfinding. The identification of Gyc76C as an essential component of the Sema-1a-PlexA repulsive axon guidance signaling pathway provides insight into how cyclic nucleotide production is linked to the cascade of events downstream of semaphorin-mediated repulsion. These observations also provide a potential target for modulating repulsive semaphorin signaling by alterations of cGMP levels directly through rGCs (Ayoob, 2004).

These analyses demonstrate a role for the rGC Gyc76C in Sema-1a-mediated axon-axon repulsion. LOF mutations were generated in the Gyc76C gene and highly penetrant phenotypes were observed similar to the motor axon guidance defects observed in sema1a, plexA, and mical mutants. Micals are a family of conserved flavoprotein oxidoreductases that function in Plexin-mediated axonal repulsion (Terman, 2002). Neuronal expression of a Gyc76C cDNA restores the wild-type innervation pattern in gyc76C mutant embryos and also restores viability to the lethal gyc76C mutant line, demonstrating a requirement for Gyc76C in neurons for correct axonal pathfinding. Neuronal overexpression of wild-type Gyc76C also results in phenotypes resembling PlexA GOF phenotypes. The genetic interaction analyses confirm a role for Gyc76C in Sema-1a-PlexA repulsive signaling. Embryos heterozygous for both Gyc76C and other members of this signaling cascade, including Sema-1a, PlexA, and MICAL, display motor axon pathway disruptions. These phenotypes are qualitatively similar to LOF mutant phenotypes observed in sema1a, plexA, and mical LOF mutants and are seen at comparable frequencies. In addition to suppressing the Sema-1a- dependent midline phenotype, loss of Gyc76C function also suppresses a PlexA- dependent phenotype. However, increasing the levels of Gyc76C enhances this PlexA GOF phenotype. Finally, a Gyc76C transgene lacking a key conserved aspartate residue required for cyclase catalytic activity does not rescue either the gyc76C embryonic motor axon guidance defects or the lethality associated with gyc76C mutants and appears to function in a dominant-negative manner. Taken together, these results link Gyc76C to the proper generation of neuromuscular connectivity in Drosophila through its role in mediating semaphorin-plexin signaling events associated with axonal repulsion. In addition, these results strongly suggest that cGMP production is critical for Gyc76C participation in Sema1a neuronal signaling events (Ayoob, 2004).

Initial in vitro observations demonstrating the importance of cGMP levels in semaphorin-mediated repulsion shows that increasing cGMP signaling reverses the repulsive signal from the secreted vertebrate semaphorin Sema3A, resulting in Sema3A acting as an attractant in the single growth cone steering assay. Recent studies show that Sema3A growth cone collapse requires increased cGMP signaling and also that cAMP signaling acts in opposition to cGMP signaling in the modulation of Sema3A-mediated growth cone collapse. Support for cAMP signaling cascades modulating semaphorin-mediated repulsion in vivo is provided by a demonstration that the A-kinase anchoring protein Nervy serves to antagonize Sema-1a-mediated axonal repulsion in Drosophila motor axons. Presumably, Nervy acts by localizing cAMP activation of PKA to the Plexin receptor and decreases Sema-1a repulsive signaling (Terman, 2004). The identification of Gyc76C as a positive effector in vivo of Sema-1a-PlexA-mediated repulsion is consistent with these Sema3A growth cone collapse studies. A model recently proposed for cyclic nucleotide modulation of netrin-1-mediated attraction and repulsion provides insight into how cGMP might effect semaphorin-mediated steering, collapse, and in vivo axonal repulsion. Using the in vitro growth cone steering assay, Nishiyama (2004) has shown that the [cAMP]/[cGMP] ratio determines whether netrin-1 acts in an attractive or a repulsive manner: high ratios promote attraction, whereas lower ratios promote repulsion. Importantly, a basal level of cGMP signaling is required for both netrin-mediated attractive and repulsive responses in this system. Although it remains to be determined, it is tempting to speculate that, like the observations for netrin-1-mediated guidance, the [cAMP]/[cGMP] ratio also serves to modulate semaphorin signaling events. In Drosophila motor axons, Gyc76C and Nervy could function antagonistically to regulate Sema-1a signaling in this manner. Gyc76C production of cGMP would lower a [cAMP]/[cGMP] ratio and thus promote repulsion, whereas increases in cAMP levels would decrease repulsion through PKA tethered to PlexA by Nervy. A loss of Gyc76C altogether would result in abolition of Sema-1a repulsion because of a cGMP requirement for any guidance response, and this is what was observed in the gyc76C mutants. Future experiments will determine how raising or lowering Gyc76C activity affects the guidance response to Sema-1a in vivo (Ayoob, 2004).

This study describes a role for a receptor-type guanylyl cyclase in axon guidance as an effector of transmembrane Sema1a axonal repulsion. Soluble guanylyl cyclases in both vertebrates and invertebrates have been implicated in axonal and dendritic guidance. However, in a GOF genetic screen for Sema-1a signaling components, genomic regions containing genes encoding all of the identified Drosophila soluble guanylyl cyclase subunits were assayed, including one known to be expressed in the nervous system, yet heterozygosity at these loci did not suppress or enhance the Sema-1a GOF phenotype. This may reflect a requirement for cGMP production at or near the PlexA receptor to provide a local increase in cGMP levels essential for semaphorin-mediated axonal repulsion and suggests that basal cGMP signaling provided by soluble gyanylyl cyclases is not essential for semaphorin-mediated repulsion. The initial genetic screen covered an additional two of the seven Drosophila rGCs, however, neither of the deficiencies that remove these rGCs genetically interacted with the Sema-1a GOF phenotype. Taken together, these results from the genetic screen suggest that Gyc76C is an integral component of the semaphorin signaling cascade and that cGMP production by other sources may not contribute to this repulsion. These results also motivate future experiments to investigate specific interactions between Gyc76C and PlexA (Ayoob, 2004).

Vertebrate receptor guanylyl cyclases that have a single transmembrane domain like Gyc76C are best known for their roles as receptors for natriuretic peptides that regulate blood pressure and volume and also for their role in the visual phototransduction cascade. The other vertebrate rGCs, however, have no known ligands or functions. In addition, very little is known about what roles, if any, these vertebrate rGCs play during neural development. It will be of great interest to investigate whether any vertebrate rGCs participate in semaphorin repulsive signaling (Ayoob, 2004).

Because Gyc76C is a multidomain protein, it is likely that regions other than the cyclase domain are important for its function. Interestingly, like the transmembrane protein Off-track, which is also required for Sema1a-mediated motor axon repulsion in Drosophila, Gyc76C contains a catalytically inactive kinase homology domain (KHD). In the vertebrate receptor guanylyl cyclase GC-A, this region has been shown to play a regulatory role by inhibiting the catalytic cyclase domain. The KHD of Gyc76C, or possibly Off-track, may function as an important modulator of cyclase activity (Ayoob, 2004).

The portion of Gyc76C that is C terminal to the conserved cyclase domain is unique among rGC family members; it is much longer than the same region in other rGCs and shares no amino acid similarity with these regions or with sequences of any known proteins. However, the last four amino acids of Gyc76C fit the consensus for a PDZ (PSD-95, Discs-large, zona occludens-1) domain binding motif. A similar motif is also found in MICAL, another component of the Sema-1a signaling cascade (Terman, 2002), raising the possibility that, as has been observed for other assemblages of signaling components, PDZ domain-containing scaffolding proteins may serve an important role in semaphorin signaling (Ayoob, 2004).

Gyc76C may provide a direct physical link between the leading edge of the growth cone and the motile machinery of the actin cytoskeleton. Vertebrate rGCs in photoreceptors are able to bind actin filaments, and the C-terminal domains of intestinal rGCs have also been implicated in interactions with the actin cytoskeleton. Perhaps the large C-terminal extension of Gyc76C functions in a similar manner to bridge the regions of signal reception and output. Whether or not Gyc76C cyclase activity is ligand gated remains unknown, and like all other Drosophila rGCs and the majority of vertebrate rGCs, Gyc76C is an orphan receptor. Future experiments will address whether Sema-1a triggers Gyc76C catalytic activity and also whether Gyc76C is indeed part of the receptor complex for Sema-1a (Ayoob, 2004).

In conclusion, using a novel genetic screening paradigm for identifying semaphorin signaling cascade components, an in vivo link was found between Sema-1a-mediated repulsive guidance and cGMP signaling pathways. Characterization of other candidates from this screen will likely provide additional insight into the mechanisms of repulsive axon guidance signaling (Ayoob, 2004).

Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions

Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. In the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. It is proposed that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons (Sweeney, 2007).

Genetic mosaic analyses of the POU transcription factor Acj6 have suggested hierarchical interactions among different classes of ORNs contribute to their axon targeting. However, it has been unclear what molecules mediate these interactions and under what cellular and developmental context these interactions take place. This study provides mechanisms to address both questions. A 'temporal target restriction' model is presented. Antennal ORN axons reach and start to pattern the developing antennal lobe before the arrival of MP axons. These early-arriving antennal axons express a high level of Sema-1a. Late-arriving MP axons express the repulsive receptor PlexinA and are repelled by Sema-1a expressed on the antennal axons. Thus, antennal ORN axons restrict MP ORN axon targeting to the proper antennal lobe region. The target glomeruli of MP classes are indeed clustered in a small area in the adult antennal lobe, surrounded by target glomeruli of antennal ORNs (Sweeney, 2007).

Multiple lines of evidence support the temporal target-restriction model. First, pioneering axons of the antennal ORNs reach the antennal lobe ~12 hr prior to those of the MP ORNs. Second, loss of antennal ORN axons results in mistargeting of MP axons, but not vice versa. Third, both Sema-1a and its known receptor PlexinA are expressed in ORN axons at appropriate developmental stages. Fourth, extensive genetic mosaic analyses of sema-1a indicate that Sema-1a is required for axon targeting of all MP ORN classes and acts non-cell-autonomously as a ligand. Fifth, knockdown of PlexinA in ORNs results in MP mistargeting phenotypes similar to those of sema-1a mosaics and those resulting from loss of antennal axons. Lastly, MP axon targeting within the antennal lobe predominantly relies on Sema-1a on antennal axons (Sweeney, 2007).

This model makes a few additional predictions that have not been directly tested due to technical limitations: (1) PlexinA should act cell autonomously in MP ORNs; (2) Sema-1a/PlexinA should mediate repulsion between antennal and MP axons; (3) the sequential arrival of antennal and MP axon innervation should be essential for their interactions. The first prediction is supported by previous findings that PlexinA acts as a receptor for Sema-1a in embryonic motor axon guidance, and PlexinA acts in ORNs and genetically interacts with Sema-1a. The second prediction is suggested by MP axon mistargeting to normal targets of antennal axons in sema-1a^−/− and plexinA RNAi conditions and is consistent with the well-documented repulsive functions of Sema-1a in Drosophila embryos and of Semaphorins more generally from insects to mammals. Finally, the temporal evidence remains correlative rather than causal, since it is currently not possible to specifically alter the sequence of axon arrival (Sweeney, 2007).

Although a central focus of this study is the axon-axon interaction between antennal and MP ORNs, it is likely that similar axon-axon interactions take place between different classes of antennal axons to regulate their targeting. The following data support this extrapolation. Antennal ORN axons express both Sema-1a and PlexinA; certain classes of antennal ORN axons require Sema-1a non-cell-autonomously; and PlexinA is required for proper targeting of many antennal ORN classes examined. A rigorous test of this extrapolation will require the identification of ORN class-specific promoters that are expressed early during development. This will allow for the examination of axon arrival timing and genetic manipulations of specific antennal ORN classes (Sweeney, 2007).

Axon-axon interactions among ORN axons likely represent one of multiple mechanisms that enable ~50 classes of ORNs to target their axons to ~50 glomeruli. In the phenotypic analyses described in this study for sema-1a and plexinA, although the severity of phenotypes varies depending on classes and genetic manipulations, the normal glomerular targets are often still innervated. This could be rationalized by the mosaic nature of sema-1a loss-of-function analyses, the partial knockdown of PlexinA by RNAi, or contributions of other ligand-receptor pairs to antenna-MP axon-axon interactions. However, even in the extreme cases of smo clones where both antennae fail to develop and all antennal axons are presumably missing, the MP axon mistargeting phenotype is only partially penetrant. These observations suggest that axon-axon interactions contribute to the fidelity of axon targeting together with other mechanisms. It is envisioned that global cues expressed in the antennal lobe act first to direct pioneering ORN axons to different general areas of the antennal lobe, axon-axon interactions then act to constrain the coarse targeting of later-arriving axons, and pre- and postsynaptic recognition contributes to the final target selection (Sweeney, 2007).

Genetic mosaic analyses indicate that Sema-1a- and PlexinA-mediated axon-axon interactions are also used among MP axons to regulate their entry into the antennal lobe. A disruption of MP-MP interactions results in occasional MP axon termination before entering the antennal lobe. This phenotype is quite analogous to the failure of motor axons to defasciculate from their fascicles upon reaching their muscle field observed in sema-1a or plexinA mutant Drosophila embryos; this embryonic phenotype has been interpreted as a defect in Sema-1a-PlexinA mediated axon-axon repulsion, which normally would facilitate defasciculation of individual axons from the rest of the fascicle. Similarly, MP-MP axon-axon repulsion mediated by Sema-1a and PlexinA may serve to loosen the individual MP axons within the bundle, allowing them to dissociate from each other and facilitate their entry into the antennal lobe (Sweeney, 2007).

A separate study shows that at an earlier stage during development, Sema-1a acts cell autonomously as a receptor (in response to an unknown ligand) in olfactory PNs for their dendritic targeting. It is thus of interest that Sema-1a acts in two different modes to regulate targeting specificity of PNs and ORNs that eventually become synaptic partners. This finding also raises the possibility that in addition to acting as a receptor for PN dendritic targeting, Sema-1a on PN dendrites might also act as a ligand for targeting of ORN axons that express and require PlexinA. However, preliminary studies have not yielded positive evidence to support this hypothesis (Sweeney, 2007).

Semaphorins and their receptors have various functions in wiring the nervous system, including olfactory systems. In mice, Semaphorin3F-Neuropilin2 signaling restricts ORN axon termination to the glomerular layer, preventing axon overshoot into deeper layers of the olfactory bulb. Moreover, Semaphorin3A-Neuropilin1 contributes to the broad organization of ORN axon targeting. Semaphorin3A, expressed in a broad compartment of the olfactory bulb by glial cells, repels Neuropilin1-expressing ORNs from this area. Sema3A-Neuropilin1 signaling has a different function in chick ORN targeting: it prevents ORNs from prematurely entering, and subsequently overshooting, the olfactory bulb. The current findings are conceptually and qualitatively distinct from these previous reports: Sema-1a mediates the interactions between axons with temporally distinct innervation patterns, rather than the interaction between axons and their targets (Sweeney, 2007).

Clear examples that temporal sequence plays an important role in neuronal wiring come from numerous studies on pioneering axons from insects to mammals. Early axons lay down the path for late ones to follow, presumably through axon-axon adhesion and fasciculation. Axon-axon interactions have also been proposed to play a role in final target selection. For example, in Drosophila photoreceptor axon targeting, R1-R6 axons from the same ommatidium, upon reaching the laminar layer, select six distinct cartridges to send their final terminal branches. Hierarchical interactions among photoreceptors contribute to their target selections, although the mechanism is unknown. In the establishment of the retinotopic map of the vertebrate visual system, relative rather than absolute EphA receptor levels on retinal ganglion cells determine the anterior-posterior positions of their axon termination at the target, likely through axon-axon interactions and competition. In mouse ORN axon targeting, axon-axon interactions have been proposed to allow ORNs expressing the same OR to converge and stabilize and to provide comparisons and discriminations of different ORN classes. The mechanisms by which these axon-axon interactions regulate targeting specificity are not well understood, and the role of temporal sequences has not been explored in these systems. A difficulty is to unravel where these neurons interact, whether at cell bodies, axon paths, or target areas. The Drosophila olfactory system provides an excellent model to explore the molecular and cellular basis of these axon-axon interactions. In particular, the physical separation of ORN cell bodies into two sensory organs, the antenna and the maxillary palp, allows assessment of afferent-afferent interactions exclusively at their final target area -- a feature exploited in this study to dissect the cellular and molecular basis of ORN axon-axon interactions (Sweeney, 2007).

Examples of a common target area innervated by multiple input axons, whether arriving simultaneously or sequentially, are ample in developing nervous systems. It is proposed that target restriction through axon-axon interactions as described here could contribute widely to establishing neuronal wiring specificity (Sweeney, 2007).

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (PlexinA and PlexinB), have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. The well-characterized sensory system of the Drosophila embryo was used to address these problems. Novel sensory axon defects were found in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes (Bates, 2007).

The detailed knowledge of the cellular basis for axon pathfinding in the sensory system of the Drosophila embryo has been exploited to shed further light on the mechanisms by which semaphorins mediate axon guidance. Analysis of sensory axon trajectories in semaphorin mutants reveals that secreted semaphorins play roles in early pathfinding decisions by these axons. In sema2a loss-of-function mutants, the v'ch1 axon often projects to a nearby, inappropriate pathway, the ISN, instead of the SN. Two other defects are seen at low frequencies in sema2a mutants: one or more of the lateral cluster axons projects aberrantly to the SN and axons of dorsal sensory neurons project anteriorly or posteriorly, instead of growing ventrally towards the lateral sensory cluster (Bates, 2007).

The modest penetrance levels of sema/plex LOF phenotypes suggest that in the sensory system, as in many other situations, axon pathfinding events are likely to involve the simultaneous action of multiple guidance factors that act cooperatively and/or redundantly. Indeed, it has been shown that PlexA and PlexB have partially redundant roles in motor axon pathfinding (Ayoob, 2006) while motor axon branching over muscles is governed by the relative balance of Sema2a, Netrins and FasII, rather than the level of any one of these molecules. Sema2a alone apparently makes only a moderate contribution to sensory axon guidance. Several other molecules have been identified that play a role in the guidance of the same sensory axons that are affected by sema and plex mutations. A future goal of this research is to elucidate how these various guidance factors interact to mediate sensory axon guidance (Bates, 2007).

Overexpression of Sema2a in oenocytes, epidermis or trachea, or ectopic expression in neurons did not result in defective sensory axon morphologies. This finding contrasts with the reported disruption of muscle innervation following overexpression of Sema2a on muscles. Using anti-Sema2a staining, it was cofirmed that the GAL4 driver lines used in these overexpression experiments led to expression of high levels of Sema2a protein in the relevant tissues. Moreover, the partial rescue of the sema2a LOF mutant phenotype observed when the sal-GAL4 line was used to drive UAS-sema2a shows that at least this line can effectively drive expression of functional Sema2a protein. Thus, the absence of sensory axon defects following Sema2a overexpression suggests that sensory axons are less sensitive to semaphorin levels than are motor axons (Bates, 2007).

In contrast to the secreted semaphorins, no evidence was found from analysis of the sema1a loss-of-function mutant and overexpression of sema1a that this transmembrane semaphorin is involved in sensory axon guidance in the periphery (Bates, 2007).

The v'ch1 pathway is a potentially attractive route for lateral cluster axons, as shown by the misprojection of lateral axons along this route in a variety of mutants, including robo, slit and trachealess. However, in wild-type embryos, lateral cluster axons very rarely follow the v'ch1 axon and vice versa. At the onset of sensory axon growth, a cluster of 4 to 7 oenocytes lies between the v'ch1 and lateral cluster neurons. Filopodia extend from the v'ch1 cell body in a variety of directions: those projecting dorsally and anteriorly, towards the oenocytes, are generally short and do not develop into axon branches. These observations suggest that oenocytes form a repulsive zone, preventing axon growth between the v'ch1 and lateral cluster cell bodies. The finding that ablating some of the oenocytes in a hemisegment can result in misprojection of the v'ch1 axon to the lateral cluster neurons and vice versa supports this hypothesis (Bates, 2007).

Oenocytes strongly express Sema2a, and driving expression of Sema2a in these cells rescues the v'ch1 axon misprojection defect seen in sema2a mutants. These findings suggest that Sema2a secreted by oenocytes normally represses axon growth by v'ch1 towards the lateral cluster neurons, and vice versa. Sema2a may act by inhibiting filopodial extension and/or by repressing filopodial dilation and subsequent axon formation. Loss of Sema2a function in sema2a mutants removes that repression, allowing the v'ch1 and lateral cluster axons to misproject in a reciprocal fashion to each other (Bates, 2007).

This model of Sema2a function differs from prevailing views of the action of secreted semaphorins as diffusible chemo-repellents. Isbister (2003) has shown that sensory axons in the grasshopper limb bud are guided by two orthogonal gradients of Sema2a protein expression which span the entire width and length of the limb bud. The directional growth of the axons is apparently dictated by the fractional change in Sema2a concentration across the limb bud epithelium. Similarly, the secreted vertebrate semaphorin Sema III is proposed to have a long-range guidance function in the spinal cord (Messersmith, 1995). This study concluded that Sema III secreted by ventral spinal cord cells diffuses dorsally, repelling the axons of small diameter sensory afferents and thereby forcing them to terminate in the dorsal horn (Bates, 2007).

In contrast, the current results suggest that the secreted Drosophila semaphorin Sema2a acts in a very local fashion. Sema2a produced by oenocytes in contact with a portion of the surface of the v'ch1 cell body suppresses axon extension specifically from that region of the cell. In this way, Sema2a determines, at least in part, the initial polarity of axon extension from the v'ch1 neuron (Bates, 2007).

Sema2a is also expressed in stripes of epidermal cells at the segment border. This source of Sema2a could conceivably contribute to guidance of the v'ch1 and lateral cluster axons by inhibiting their growth in a posterior direction. However, the absence of aberrant, posteriorly projecting sensory axons on either v'ch1 or lateral cluster neurons in sema2a mutants speaks against this function (Bates, 2007).

Genetic and biochemical evidence points to PlexA being the receptor for the transmembrane class I semaphorins during motor axon guidance in the Drosophila embryo. However, until recently, the receptor(s) for the secreted class II semaphorins and the ligand(s) for PlexB had not been identified. Ayoob (2006) has now provided evidence for a physical and genetic interaction between PlexB and Sema2a during motor axon guidance (Bates, 2007).

The current study provides a number of lines of evidence, suggesting that PlexB acts as a receptor for Sema2a in a different context, regulating the growth of the v'ch1 sensory axon. (1) plexB is expressed in lateral cluster sensory neurons, including v'ch1 at the time of axon outgrowth. (2) plexB loss-of-function mutants show the same v'ch1 axon misprojection phenotypes as sema2a loss-of-function mutants. (3) The v'ch1 axon defects in plexB mutants can be rescued by driving plexB expression in sensory neurons. One caveat with this conclusion is that the P0163-GAL4 line used in these experiments drives GAL4 in the oenocytes as well as the sensory neurons. (4) Halving the gene dose of both sema2a and plexB results in the same v'ch1 axon defects as seen in single sema2a or plexB homozygous mutant embryos (Bates, 2007).

A parallel set of data leads to the tentative suggestion that PlexA may act as a receptor for Sema2a during lateral cluster axon guidance: plexA is expressed in lateral cluster neurons; plexA and sema2a LOF mutants show defects in lateral cluster axon growth, albeit at low penetrance levels; these defects can be rescued by driving PlexA in the sensory neurons; and sema2a^−/+; plexA^−/+ embryos show the same lateral cluster axon defects (Bates, 2007).

While the above results provide support for the idea that sensory axon guidance is mediated by Sema2a signaling through both PlexA and PlexB receptors, the mechanism is likely to be more complex than binding of Sema2a to PlexA and PlexB and consequent independent activation of these two receptors. The increased penetrance of the v'ch1 defect in double homozygous sema2a; plexA and sema2a; plexB mutants, compared to single sema2a, plexA or plexB mutants, suggests that additional ligands are involved. Direct interactions between the PlexA and PlexB receptors, as demonstrated by the co-immuno-precipitation experiments of (Ayoob, 2006), may also contribute to the increased frequency of v'ch1 axon defects observed in double sema2a; plexA and sema2a; plexB mutants compared to single mutants. It is believed that the sensory system provides a valuable platform in which to further investigate this and other issues related to semaphorin function in axon guidance (Bates, 2007).

Highwire regulates guidance of sister axons in the Drosophila mushroom body

Axons often form synaptic contacts with multiple targets by extending branches along different paths. PHR (Pam/Highwire/RPM-1) family ubiquitin ligases are important regulators of axon development, with roles in axon outgrowth, target selection, and synapse formation. This study reports the function of Highwire, the Drosophila member of the PHR family, in promoting the segregation of sister axons during mushroom body (MB) formation. Loss of highwire results in abnormal development of the axonal lobes in the MB, leading to thinned and shortened lobes. The highwire defect is attributable to guidance errors after axon branching, in which sister axons that should target different lobes instead extend together into the same lobe. The highwire mutant MB displays elevation in the level of the MAPKKK Wallenda/DLK (dual leucine zipper kinase), a previously identified substrate of Highwire, and genetic suppression studies show that Wallenda/DLK is required for the highwire MB phenotype. The highwire lobe defect is limited to α/β lobe axons, but transgenic expression of highwire in the pioneering α'/β' neurons rescues the phenotype. Mosaic analysis further shows that α/β axons of highwire mutant clones develop normally, demonstrating a non-cell-autonomous role of Highwire for axon guidance. Genetic interaction studies suggest that Highwire and Plexin A signals may interact to regulate normal morphogenesis of α/β axons (Shin, 2011).

In Drosophila, highwire is best studied for its role in restraining synaptic terminal growth at the NMJ. Studies in the fly neuromuscular system did not, however, find a role for highwire in motoneuron axon guidance. The current study demonstrates that highwire does regulate axon guidance of MB neurons in Drosophila. The gross morphological defects, such as the short α lobe and thinning of either the α or β lobe, present in the highwire MB lobes of the adult could be attributable to defects in either the development or maintenance of axons. However, similar defects were observed in both the developing and adult MB, so the phenotype is not attributable to degeneration of previously formed axons. The defect is also inconsistent with a gross alteration in axon outgrowth or guidance. The α/β axons form, path-find appropriately through the peduncle, and branch at the appropriate location. Instead, the data suggest a selective deficit in responding to guidance cues at this choice point. After bifurcation of the axon, sister branches do not segregate into distinct lobes as in WT but rather travel together into the same lobe. This phenotype is consistent with loss of homotypic repulsion of sister branches and/or the inability to respond to selective guidance cues targeting the axons to particular lobes (Shin, 2011).

In both fly and worm, PHR proteins sculpt synaptic terminals by restraining Wnd/DLK MAPKKK activity. In Drosophila, highwire acts as an ubiquitin ligase to limit the abundance of Wnd/DLK. Excess Wnd/DLK protein overactivates a MAP kinase signaling pathway that promotes synaptic terminal overgrowth. This study demonstrates that highwire-dependent downregulation of Wnd/DLK is also required for segregation of sister branches of α/β axons and, hence, proper MB development. In the absence of highwire, levels of Wnd/DLK are elevated in the axons of the developing MB. Furthermore, genetic deletion of Wnd/DLK suppresses the highwire-dependent phenotypes, demonstrating that Wnd/DLK is required for the aberrant behavior of α/β axons in the highwire mutant. Attempted were made to test whether overexpression of Wnd/DLK phenocopies highwire mutant MB by driving Wnd/DLK transgenic expression with OK107-Gal4 or MB subset Gal4 lines, including α'/β'-specific NP2748-Gal4. However, the strong overexpression of Wnd/DLK resulted in either lethality or massive cell death in the MB, probably because of the excess activation of downstream JNK MAPK signaling (Shin, 2011).

Although the relationship between the PHR ubiquitin ligase and DLK kinase is clear in flies and worms, studies in vertebrate systems paint a murkier picture. Analysis of PHR mutants in mice and zebrafish consistently demonstrate an important role in various aspects of axon development. However, the molecular mechanism of PHR action and the potential involvement of DLK in vertebrate axons is less clear. In cultured sensory axons from the Phr1 mutant magellan, axon morphology is disrupted and DLK protein is mislocalized. In addition, pharmacological inhibition of p38, a MAP kinase that can be downstream of DLK, reduced the size of the abnormally large growth cones present in these mutant axons. These findings are consistent with a role for DLK activity in generating the Phr1-dependent phenotypes. However, in an independently generated Phr1 mutant, no gross change was observed in DLK levels and it was found that genetic deletion of DLK failed to suppress either corticothalamic axon guidance defects or motoneuron sprouting defects. In zebrafish, mutations in the PHR ortholog esrom disrupt axon guidance and lead to an increase in JNK activation, consistent with a role for DLK, but inhibition of neither JNK nor p38 can suppress the esrom phenotypes, arguing against a functional role for DLK (Hendricks, 2009). The finding that Wnd/DLK is essential for axonal phenotypes in Drosophila while it is dispensable for at least some axonal phenotypes in mice and fish suggests that there is no simple relationship between PHR targets and the cellular function of PHR proteins. PHR proteins do interact with a number of other proteins besides DLK, and so the mechanism of PHR-dependent axonal phenotypes is likely context dependent (Shin, 2011).

During MB development, the α/β neurons are the last to be born and the last to extend their axons into the MB lobes. These α/β axons follow the path established by the earlier-born γ and α'/β' axons. In the highwire mutant, the α/β axons form short, thin, or absent α/β lobes, whereas the γ and α'/β' axons form morphologically normal lobes. Although such results would be consistent with a unique requirement for highwire in α/β neurons, a series of findings instead indicate that highwire is required in α'/β' neurons and indirectly affects the development of α/β axons via a non-cell-autonomous mechanism. First, in the highwire mutant, Wnd/DLK levels are elevated in γ, α'/β', and α/β axons, demonstrating that the Highwire ligase is likely targeting Wnd/DLK in all three cell types. Second, in the highwire mutant, the sister branches of the α/β axons fail to segregate but instead travel into the same lobe. However, within a brain hemisphere, most of the sister branches choose the same lobe, resulting in either a thickened α or β lobe. Hence, the decision as to which lobe to enter is apparently not determined independently by each axon. Third, expression of highwire in the earlier-born α'/β' neurons is sufficient to rescue the defects in the α/β lobes. Fourth, in single-cell highwire α/β clones, sister axons segregate normally in an otherwise heterozygous background. Together, these data demonstrate a non-cell-autonomous requirement for highwire (Shin, 2011).

How might α'/β' axons affect the guidance decision of α/β axons? Misexpression of the cell adhesion molecule Fas II in the α'/β' neurons leads to the loss of either α or β projections, demonstrating that inter-axonal interactions can affect α/β axon development and suggesting that α'/β' axons act as “pioneering axons” for the later-arriving α/β axons. Because the α'/β' axons form morphologically normal lobes in the highwire mutant, the defect is likely at the molecular level, potentially involving a change in either membrane-associated or secreted guidance cues. In the vertebrate CNS, the highwire ortholog Phr1 is also required for a non-cell-autonomous mechanism that guides cortical axons. In the absence of Phr1, cortical axons stall at the corticostriatal border and do not contribute to the internal capsule. In contrast, after conditional excision of Phr1 exclusively in cortical neurons, these same cortical axons can now cross this choice point and path-find to the thalamus. Hence, the requirement for PHR proteins for non-cell-autonomous axon guidance mechanisms is evolutionarily conserved, although there is no evidence that the molecular mechanism is conserved (Shin, 2011).

To investigate the molecular mechanism of the non-cell-autonomous requirement for highwire, genetic interactions between highwire and candidate guidance molecules were tested. The data suggest that Highwire promotes a Plexin A signaling mechanism that is required for proper α/β lobe development. Loss of a single copy of the plexin A gene has no effect on MB development in an otherwise WT background but enhances the phenotype of a weak highwire allele. Furthermore, RNAi-mediated knockdown of plexin A in the MB has a very similar phenotype to loss of highwire, with abnormal thickness of α/β lobes and shortened α lobes. Hence, Plexin A is required for normal MB development. Plexins are receptors for semaphorins, and both Sema-1a and Sema-5c are required for normal MB development. The genetic studies did not uncover a genetic interaction between either of these semaphorins and highwire, but the absence of such an interaction does not rule out the involvement of these or other semaphorins. Two potential models are consistent with these genetic interaction studies. First, Plexin A may function to downregulate Wnd/DLK, potentially via inhibition of Rac GTPase signaling. In the absence of either plexin A or highwire, Wnd/DLK activity would be upregulated, disrupting axonal interactions between α'/β' axons and α/β axons via unknown mechanisms. Alternatively, excess Wnd/DLK activity in the highwire mutant could disrupt the Plexin A signaling pathway that is necessary for α/β lobe development. The mechanisms by which Highwire and Plexin A signaling converge will be the subject of future studies (Shin, 2011).

EVOLUTIONARY HOMOLOGS

Drosophila Plexin B

Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, a physical map of this chromosome is being assembled. The sequence of two cosmids representing approximately 5% of the polytenized region is reported here. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis. These repeated sequences are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus gene and two new genes: one with a predicted amino acid sequence similar to ribosomal protein S3a, which is consistent with the Minute(4)101 locus thought to be in the region, and a second, plexinB, a novel member of the protein family that includes mammalian plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome (Locke, 1999).

Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling

Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. A seven amino acid sequence in PlexB is required for Rac^GTP binding. The interaction of PlexB with Rac^GTP is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters Rac^GTP from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to Rac^GTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA (Hu, 2001).

Plexin B binds to the active form of Rac (Rac^GTP); the binding maps to a 147 amino acid region, PlexBDelta3 (amino acids 1617 through 1765). To identify the critical binding sequence in PlexBDelta3, small deletions and point mutations were introduced and a seven amino acid sequence NTLAHYG (1722 through 1728) toward the C terminus of PlexBDelta3 has been identified that, when deleted, abolishes Rac binding (PlexBDelta3d7). Deletions in neighboring regions 1743 through 1759 (PlexBDelta3d17) and 1707 through 1714 do not affect Rac binding (Hu, 2001).

The NTLAHYG sequence is highly conserved among Plexin family members. In particular, the tyrosine residue within the sequence is invariable. In human Plexin B1, a putative Cdc42/Rac interactive binding (CRIB)-like motif right after this conserved sequence has been described. Although the CRIB-like motif is not found in Drosophila PlexB, this may reflect a conservation of the binding mechanism at a higher structural level. The Psi blast program predicts two blocks of sequences in the Plexin cytoplasmic domain that share similarity with R-ras family GAP proteins. The sequence needed for Rac^GTP binding is located between these two GAP-like regions (Hu, 2001).

In the Drosophila genome, there are six Rho family small GTPases: Rac1 (referred to here as Rac), Rac2, Cdc42, RhoA, Mtl, and RhoL. To gain some insight into the specificity of the interaction, the binding of PlexBDelta3 with all six Drosophila Rho-like GTPases was examined. Only Rac and Rac2, which share the highest degree of sequence similarity (93% identity), show strong interactions with the BDelta3 region of PlexB (Hu, 2001).

Several lines of evidence suggest that RhoA is also involved in PlexiB signaling. Clustering of the vertebrate PlexB in Swiss 3T3 cells leads to stress fiber formation, indicative of Rho activation. The response can be blocked by inhibitors of Rho or of its downstream effector Rho kinase. Genetic data also indicate that RhoA mediates part of Plexin B signaling in embryonic axon guidance. It was of interest, then, to enquire whether RhoA may also directly associate with PlexB (Hu, 2001).

PlexBDelta, a larger piece of the PlexB cytoplasmic domain (1617 through 1827) binds to RhoA. In contrast to a preferential binding to GTPgammaS-bound Rac, PlexBDelta binds to the GTPgammaS and GDP-bound forms of RhoA equally well. The binding requires the last 40 amino acids of PlexBDelta. The seven amino acid internal deletion that eliminates PlexBDelta binding to Rac does not affect its binding to RhoA. Thus, two independent regions in PlexB cytoplasmic domain have been defined that are important for PlexB association with Rac and RhoA, respectively. Cdc42, another Rho family GTPase, does not bind to PlexBDelta (Hu, 2001).

Dosage-sensitive genetic interactions suggest Rac antagonizes Plexin B signaling. Since there is no mutant available for PlexB with which to examine genetic interactions, whether the gain of function phenotype of PlexB is sensitive to the level of expression of Rac or RhoA was examined. PlexB is endogenously expressed by CNS neurons. Overexpression of PlexB in all embryonic CNS neurons can be achieved with the UAS-GAL4 binary expression system. Flies containing the UAS-PlexB transgene reporter are crossed to flies carrying a neuron-specific transcriptional control driver, elav-GAL4. With two independent UAS-PlexB transgenic lines, a consistent, GAL4-dependent phenotype was observed in specific motor nerve branches. In particular, a striking defect was observed in the ability of the ISNb (intersegmental nerve b) motor axons to innervate the ventral longitudinal muscles 7, 6, 13, and 12. In wild-type embryos, the ISNb projects into the ventral longitudinal muscles. Particular motor axons innervate specific muscles; for example, the RP3 motor axon innervates muscles 7 and 6, while other ISNb axons innervate muscles 13 and 12. When PlexB is overexpressed in these neurons, two types of phenotypes are observed that are consistent with PlexB being a repulsive guidance receptor for muscle-expressed Semaphorins: (1) the RP3 axon frequently fails to defasciculate from the ISNb motor nerve branch, and as a result muscles 7 and 6 are uninnervated; (2) ISNb axons often fail to reach their distal-most target muscle 12 (scored as 'stall'). The copy number of UAS-PlexB transgene and elav-GAL4 driver was varied to generate embryos with a range of levels of expression of PlexB; 'RP3 missing' and 'stall' phenotypes are dose dependent. This dosage sensitivity suggests that the PlexB gain-of-function phenotype may provide a sensitive background for revealing genetic interactions with genes encoding downstream components involved in Plexin B signaling (Hu, 2001).

Does the binding of PlexB to Rac increase or decrease the output of Rac? It was reasoned that if increasing PlexB expression produces its effect by activating Rac, then genetically limiting Rac gene dose might suppress the PlexB overexpression phenotype. Alternatively, if PlexB signals by turning down Rac activity, then an enhancement of the plexB overexpression phenotypes might result when Rac is reduced. Indeed, the results support the second alternative: PlexB inactivates Rac. Rac protein level was reduced by 50% using a small deficiency line, Df(3L)Ar14-8 (61C04-62A08), in a moderate PlexB overexpression background (one copy transgene, one copy driver). This resulted in a distinct increase in the penetrance of PlexB gain of function phenotypes. A complementary effect results when Rac dosage is increased in the same neurons where PlexB is overexpressed with a UAS-Rac transgene. Under such conditions, a suppression on the PlexB gain of function phenotypes is observed (Hu, 2001).

Consistent with the idea that PlexB signals by downregulating Rac activity, the Plexin gain-of-function stall phenotype is reminiscent of the loss-of-function phenotype of a positive regulator of Rac, the Trio GEF. Trio has been shown to play a role in axon guidance in Drosophila and nematode and has provided additional evidence that, in this capacity, Trio interacts with Rac and regulates PAK activity. Similar to reducing Rac, reducing Trio enhances PlexB gain-of-function phenotype. However, the enhancement caused by reducing Trio is not as great as that caused by reducing Rac. This probably reflects that Trio is not directly coupled to PlexB and is not the only positive regulator (GEF) for Rac in motor axons. Rather, Trio is likely to be one of many positive regulators of Rac in these axons (Hu, 2001).

The role of RhoA in PlexB signaling was examined by reducing RhoA gene dosage with two different RhoA mutant alleles, Rho^rev220 and RhoA^l(2)k07236. Instead of enhancing the PlexB gain-of-function phenotypes as the Rac deficiency does, partially removing RhoA suppresses the PlexB gain-of-function phenotypes. This result suggests that RhoA acts antagonistically to Rac and, moreover, that RhoA partially mediates Plexin B signaling (Hu, 2001).

To further test the model that PlexB downregulates Rac output, the effect of increasing Plexin was examined in Rac dominant-negative embryos. No mutant for Drosophila Rac has yet been published, but a loss-of-function analysis for Rac, achieved by overexpressing a dominant-negative form of Drac (N17Rac) in neurons, has revealed dramatic defects in motor axon guidance. The same ISNb nerve branch that is affected by PlexB overexpression is also sensitive to overexpression of dominant-negative Rac (N17Rac). The predominant ISNb defect in N17Rac embryos occurs at an earlier target entry point, where the whole ISNb branch normally branches off from ISN nerve. In N17Rac embryos, the ISNb fails to enter the ventral muscles and instead follows the ISN distally toward dorsal muscles (scored as 'bypass'). The difference in the quality of the ISNb phenotype of N17Rac and Plexin B gain of function embryos may likely reflect the fact that Rac is downstream of multiple guidance receptors (Hu, 2001).

The penetrance of the N17Rac bypass phenotype is very sensitive to gene dosage. When the N17Rac transgene is expressed using drivers of different strengths, different frequencies of defects result. This suggests that N17Rac only partially knocks out the wild-type gene function and that expressing N17Rac with a driver of medium strength may provide a sensitized background for testing genes that regulate the remaining Rac activity. It was reasoned that if the Plexin and Rac interaction regulates Rac activity, then it might be possible to alter the penetrance of the N17Rac bypass phenotype by simultaneously increasing PlexB gene dose in the same neurons. Indeed, coexpressing PlexB and RacN17 results in a distinct enhancement of the ISNb bypass phenotypes. N17Rac embryos also show bypass defects in the SNa motor axons that project to lateral muscle targets. This SNa bypass phenotype has never been observed in any other mutant background, and it also turns out to be enhanced by simultaneously overexpressing PlexB in these neurons (Hu, 2001).

In a reciprocal experiment PlexB was reduced in Rac dominant-negative embryos to see if this had an opposite effect. This was done by injecting double-strand RNA of PlexB into N17Rac embryos. N17Rac embryos injected with Plexin B dsRNA show distinct reduction in bypass defects compared with N17Rac embryos injected with buffer. Thus, reducing PlexB and increasing PlexB in Rac dominant embryos produces opposite modulations, consistent with the model that PlexB downregulates Rac activity (Hu, 2001).

To test whether the PlexB gain-of-function and the genetic interactions depend on the direct association between PlexB and Rac, a mutant PlexB transgene, UAS-Plex Bd7, was constructed containing the seven amino acid NTLAHYG deletion in the Rac binding region of an otherwise wild-type PlexB. The same enhancement test on N17Rac embryos was performed with this mutant transgene, and no enhancement was observed. The PlexB gain-of-function phenotypes also seem to be dependent on this Rac binding region. In contrast to wild-type PlexB, when the d7 mutant transgene is overexpressed under the control of the same neuronal GAL4 driver elav, the frequency of ISNb phenotypes is significantly lower. This low-penetrance phenotype is not enhanced by removing one copy of Rac (Hu, 2001).

In vivo expression and targeting of PlexBd7 transgene could not be examined due to the lack of PlexB antibody. Nevertheless, the seven amino acid deletion does not affect protein expression and stability of PlexBDelta3 in in vitro experiments. Three independent lines of PlexBd7 transgenes show consistent behavior when tested for their phenotypes and interactions with Rac, arguing that the negative result is not caused by the insertion site (Hu, 2001).

In light of the genetic interactions between PlexB and Rac, the biochemical nature of this negative regulation was investigated. PlexB was found to compete with the Rac downstream effector PAK (p21-activated kinase) for binding to Rac^GTP. PAK is a serine/threonine kinase that mediates a major part of Rac signaling output to actin polymerization. Upon binding to Rac^GTP, PAK undergoes a conformational change that releases an autoinhibition on the kinase domain and becomes active. Since Rac binding is critical for PAK activation and also because PlexB and PAK bind to Rac in the same GTP-dependent manner, it was asked whether PlexB and PAK may bind to the same region of Rac and whether their binding to Rac^GTP is mutually exclusive (Hu, 2001).

An in vitro pull-down competition assay was used in which in vitro translated L61Rac was incubated with bead-bound GST-PAK^1-141 in the presence or absence of soluble PlexB protein fragment: PlexBDelta2(1619-1753). (PlexBDelta2 is a 135 amino acid fragment of PlexB. It binds to Rac^GTP equally well as PlexBDelta3, but it can be expressed at a higher level.) When Plexin BDelta2 is present in the binding solution, the amount of RacL61 pulled down by PAK^1-141 is greatly reduced. The extent of reduction is dependent on the amount of PlexBDelta2 used. At the 48:1 molar ratio of PlexBDelta2 to PAK^1-141, the reduction is close to complete. PlexBDelta2d7, a deletion PlexB fragment that is incapable of binding to Rac, does not compete with PAK for the Rac binding. Conversely, the presence of PAK protein fragment PAK^78-151 also reduces RacL61 binding to PlexBDelta3. This shows that the binding of the two proteins to Rac^GTP is indeed mutually exclusive (Hu, 2001).

Does this competition exist in vivo? If it does, then it may be expected that overexpressing PAK together with PlexB in embryos will cancel out PlexB gain-of-function effect. Indeed, overexpressing PAK in a PlexB gain-of-function background suppresses the phenotypes of the latter, demonstrating that PlexB signaling can be antagonized by the Rac effector PAK in vivo (Hu, 2001).

It is concluded that PlexB mediates repulsion in vivo in part by binding to active Rac (Rac^GTP) and downregulating its effector output and in part by binding to and activating RhoA. Biochemical analysis shows that PlexB binds to Rac^GTP. A seven amino acid sequence in the cytoplasmic domain of PlexB is required for this binding. Genetic analysis shows that PlexB downregulates the output of Rac^GTP. Removal of one copy of Rac enhances a PlexB gain-of-function phenotype, while overexpression of PlexB enhances a Rac dominant-negative phenotype in motor axon guidance. Overexpression of a mutant form of PlexB that lacks the seven amino acid sequence required for Rac binding does not generate its own gain-of-function phenotype, and it does not enhance a Rac dominant-negative phenotype. It is also shown that PlexB binds to RhoA through a different region of its cytoplasmic domain. Although the biochemical mechanism is not known, genetic analysis suggests that PlexB increases the output of RhoA (Hu, 2001).

The results presented here allow a confirmation and extension of a current model concerning the role of GTPases in axon guidance. This model suggests that attractive guidance cues locally activate Rac or Cdc42 in the growth cone while repulsive guidance cues locally activate RhoA. It is argued that what is important is the relative balance in the output of Rac versus RhoA. An example is provided in which the PlexiB receptor mediates repulsive axon guidance by downregulating Rac^GTP output and simultaneously upregulating RhoA output. A coordinate regulation of these two small GTPases may allow the receptor to have a finer control over actin regulatory machinery. Semaphorin signaling can be converted from repulsion to attraction by changes in cGMP level. It would be interesting to test whether and how the cGMP signaling can affect this Rac/Rho balance (Hu, 2001).

Drosophila has two Plexins: A and B. Both Plexin A and B are highly expressed in the central nervous system. The two proteins share high sequence similarity in their cytoplasmic domain, indicating a similar mode of signaling shared by the two. A direct physical association of Rac^GTP with PlexB but not with PlexA has been demonstrated. However, genetic interactions have been found between Rac and both Plexins. For example, increasing PlexA also enhances the Rac dominant-negative phenotype as does PlexB. In COS cell and DRG neurons, Rac shows coclustering with PlexA upon Sema3A ligand treatment. It is likely that ligand binding to PlexA causes Rac binding (and subsequent inactivation of Rac) just as with PlexB, but it may be that PlexA requires an unknown third protein to help mediate or facilitate this physical interaction. From a genetic perspective, they both appear to function in the same way, mediating repulsion at least in part by inactivating Rac (Hu, 2001).

The transmembrane protein OTK associates with Plexin A and contributes to the Sema 1a/Plexin A signaling pathway. Mammalian Plexin B1 also coimmunoprecipitates with OTK. In the future, it will be interesting to test whether PlexB also interacts with OTK in vivo and to what degree the Rac/Rho GTPases and OTK signaling pathways function together or in parallel downstream of Plexins (Hu, 2001).

Plexin structure

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. This study presents the 2.4 A crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120(GAP).H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, it was shown that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation (Tong, 2009).

Plexin cell surface receptors bind to semaphorin ligands and transduce signals for regulating neuronal axon guidance. The intracellular region of plexins is essential for signaling and contains a R-Ras/M-Ras GTPase activating protein (GAP) domain that is divided into two segments by a Rho GTPase-binding domain (RBD). The regulation mechanisms for plexin remain elusive, although it is known that activation requires both binding of semaphorin to the extracellular region and a Rho-family GTPase (Rac1 or Rnd1) to the RBD. This study reports the crystal structure of the plexin A3 intracellular region. The structure shows that the N- and C-terminal portions of the GAP homologous regions together form a GAP domain with an overall fold similar to other Ras GAPs. However, the plexin GAP domain adopts a closed conformation and cannot accommodate R-Ras/M-Ras in its substrate-binding site, providing a structural basis for the autoinhibited state of plexins. A comparison with the plexin B1 RBD/Rnd1 complex structure suggests that Rnd1 binding alone does not induce a conformational change in plexin, explaining the requirement of both semaphorin and a Rho GTPase for activation. The structure also identifies an N-terminal segment that is important for regulation. Both the N-terminal segment and the RBD make extensive interactions with the GAP domain, suggesting the presence of an allosteric network connecting these three domains that integrates semaphorin and Rho GTPase signals to activate the GAP. The importance of these interactions in plexin signaling is shown by both cell-based and in vivo axon guidance assays (He, 2009).

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. This study reports the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation (Nogi, 2010).

C. elegans plexin

The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos, including axonal guidance. Putative null mutants of the C. elegans plexinA gene, plx-1, have been generated and characterized. plx-1 mutants exhibit morphological defects: displacement of ray 1 and discontinuous alae. The epidermal precursors for the affected organs are aberrantly arranged in the mutants, and a plx-1::gfp transgene is expressed in these epidermal precursor cells as they undergo dynamic morphological changes. Suppression of C. elegans transmembrane semaphorins, Ce-Sema-1a and Ce-Sema-1b, by RNA interference causes a displacement of ray 1 similar to that of plx-1 mutants, whereas mutants for the Ce-Sema-2a/mab-20 gene, which encodes a secreted-type semaphorin, exhibits phenotypes distinct from those of plx-1 mutants. A heterologous expression system has shown that Ce-Sema-1a, but not Ce-Sema-2a, physically binds to PLX-1. These results indicate that PLX-1 functions as a receptor for transmembrane-type semaphorins, and, though Ce-Sema-2a and PLX-1 both play roles in the regulation of cellular morphology during epidermal morphogenesis, they function rather independently (Fujii, 2002).

Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes a divergent axon guiding TGF-β, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. A model is presented for the integrated control of TGF-β, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail (Ikegami, 2004).

The male tail in C. elegans is characterized by nine distinct, linearly arranged sensory rays visible as finger-like protrusions on each side of the animal. Each ray comprises three cells that derive from a common ray precursor cell (Rn cell where n = 1-9). Each Rn cell divides to form Rn.p, a hypodermal cell, and Rn.a. The Rn.a descendants form a pre-ray cluster of three lineally related cells that eventually form two neurons (RnA and RnB) and a structural support cell (Rnst), which encases the sensory endings of these neurons to form an adult ray. Initially, the Rn.a descendants (3-cell cluster) of one ray contact the Rn.a descendants destined to form neighboring rays. They then undergo dynamic shape and position changes that separate each cluster of three lineally related cells from neighboring 3-cell clusters to ultimately form the distinct ray sensillae of the adult male. These changes are orchestrated by active cellular mechanisms involving specific cell-cell interactions within and between the 3-cell clusters destined to form rays. The final positions of the adult rays are determined by the site of attachment of the structural support cell Rnst (one of the Rn.a descendants in each cluster) to the basal surface of the cuticle in L4 male larvae (Ikegami, 2004 and references therein).

In mutants of mab-20, neighboring Rn.a descendants for each ray frequently fail to separate from each other, resulting in fused rays. Mutants of ephrin-4 (efn-4/mab-26) are known to share a similar male tail phenotype with mutants of mab-20. efn-4 encodes an ephrin-related protein that fails to bind to VAB-1, the only known ephrin receptor in C. elegans. As shown by the nonadditive ray fusion defects of mab-20 and efn-4 null mutants, it was proposed that these two genes function in a common pathway to sort the 3-cell clusters destined to form individual rays. The functional relatedness of efn-4 to mab-20 suggests the existence of crosstalk and a possible ancestral link between ephrin and semaphorin signaling mechanisms (Ikegami, 2004 and references therein).

The fact that only mutants of mab-20/sema-2A and efn-4/mab-26 were identified in large-scale screens for male ray fusion defects suggests that the signaling mechanisms that are regulated by MAB-20/Sema-2A and EFN-4/MAB-26 are encoded largely by genes that are either essential for viability or are redundant. Modifier screens have proven to be especially useful for revealing such genes. Therefore, in order to identify novel semaphorin signaling pathways and novel components of known semaphorin signaling pathways, and to better understand how ephrin and semaphorin signaling are integrated, a large-scale screen was undertaken for mutations that enhance a weak allele of mab-20. This screen identified mutants of the C. elegans plexin-2 (plx-2) gene, which encodes a presumed receptor for Sema-2A/MAB-20, plus mutants of at least six erf genes (erf-1 to erf-5 plus plx-2) that enhance both the body morphology and male ray fusion defects of mab-20(bx61^ts). unc-129, which encodes a TGF-β that regulates axon guidance in C. elegans, was also found to enhance ray fusion defects of a mab-20 weak allele. Genetic and phenotypic characterization of these mutants has begun to reveal hierarchical pathways of semaphorin and ephrin function involved in several aspects of C. elegans morphogenesis. For example, even though putative null alleles of plx-2 do not cause a male ray fusion phenotype resembling the mab-20 mutants, genetic interactions between plx-2, efn-4, unc-129, and erf mutations have revealed the existence of redundant mechanisms that regulate the signaling of mab-20 and has suggested a role (in the context of a functional network) for efn-4, plx-2, and unc-129 in integrating these pathways. The integrated regulation of semaphorin, ephrin, and TGF-β signaling in the sorting of Rn.a descendants into distinct rays in C. elegans may have implications for regulating differential cell adhesions involved in a variety of cell movements and cell shape changes that occur during animal development (Ikegami, 2004).

Plexins are functional receptors for Semaphorin axon guidance cues. Previous studies have established that some Plexins directly bind RAC^GTP and RHO. Recent work in C. elegans has shown that semaphorin 1 (smp-1 and smp-2) and plexin 1 (plx-1) are required to prevent anterior displacement of the ray 1 cells in the male tail. plx-1 is shown genetically to be part of the same functional pathway as smp-1 and smp-2 for male ray positioning. RAC GTPase genes mig-2 and ced-10 probably function redundantly, whereas unc-73, which encodes a GEF for both of these GTPases, is required cell autonomously for preventing anterior displacement of ray 1 cells. RNAi analysis indicates that rho-1-encoded RHO GTPase, plus let-502 and K08B12.5-encoded RHO-kinases, are also required to prevent anterior displacement of ray 1 cells, suggesting that different kinds of RHO-family GTPases act similarly in ray 1 positioning. At low doses of wild-type mig-2 and ced-10, the Semaphorin 1 proteins no longer act through PLX-1 to prevent anterior displacements of ray 1, but have the opposite effect, acting through PLX-1 to mediate anterior displacements of ray 1. These results suggest that Plexin 1 senses levels of distinct RHO and RAC GTPases. At normal levels of RHO and RAC, Semaphorin 1 proteins and PLX-1 prevent a forward displacement of ray 1 cells, whereas at low levels of cycling RAC, Semaphorin 1 proteins and PLX-1 actively mediate their anterior displacement. Endogenously and ectopically expressed SMP-1 and SMP-2 suggest that the hook, a major source of Semaphorin 1 proteins in the male tail, normally attracts PLX-1-expressing ray 1 cells (Dalpé, 2004).

Vulva development in C. elegans involves cell fate specification followed by a morphogenesis phase in which homologous mirror image pairs within a linear array of primordial vulva cells form a crescent shape as they move sequentially towards a midline position within the array. The homologous pairs from opposite half vulvae in fixed sequence fuse with one another at their leading tips to form ring-shaped (toroidal) cells stacked in precise alignment one atop the other. The semaphorin 1a SMP-1, and its plexin receptor PLX-1, are required for the movement of homologous pairs of vulva cells towards this midline position. SMP-1 is upregulated on the lumen membrane of each primordial vulva cell as it enters the forming vulva and apparently attracts the next flanking homologous PLX-1-expressing vulva cells towards the lumen surface of the ring. Consequently, a new ring-shaped cell forms immediately ventral to the previously formed ring. This smp-1- and plx-1-dependent process repeats until seven rings are stacked along the dorsoventral axis, creating a common vulva lumen. Ectopic expression of SMP-1 suggests it has an instructive role in vulva cell migration. At least two parallel acting pathways are required for vulva formation: one requires SMP-1, PLX-1 and CED-10; and another requires the MIG-2 Rac GTPase and its putative activator UNC-73 (Dalpe, 2005).

Mutation of zebrafish plexin

One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis it was shown that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, sidetracked (plexin A3) is shown to play an additional role in motor axonal morphogenesis. Together, these data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. It is proposed that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance (Palaisa, 2007).

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord (Tanaka, 2007).

Plexins: receptors for semaphorins

Immunohistochemistry, using monoclonal antibodies (named A5 and B2) that specifically recognize cell surface proteins neuropilin and plexin, respectively, has revealed that olfactory axons in Xenopus tadpoles can be classified into several subgroups by virtue of the expression levels of these two cell surface molecules. The vomeronasal axons express plexin but not neuropilin. The plexin-positive and neuropilin-negative vomeronasal axons form a discrete fiber bundle, even after they join with the principal olfactory axons. However, the principal olfactory axons can divided into at least two subclasses; the neuropilin-predominant axons, which express high levels of the neuropilin and low levels of the plexin, and the plexin-predominant axons, which express high levels of the plexin and low levels of the neuropilin. Within the olfactory nerve the pathways for these two principal olfactory axon subclasses are initially intermingled with one another, but gradually segregate throughout their courses from the nose to the cerebrum. Eventually, the neuropilin-predominant and the plexin-predominant principal olfactory axon subclasses project to specified glomeruli in topographically related regions within the main olfactory bulb. Neuroanatomical tracings of the olfactory projection also confirm the gradual segregation of the pathways for the principal olfactory axons. These results allow for the speculation that both the neuropilin and the plexin are involved in axon interactions, and play roles in the organization of the precise patterns of the olfactory pathway and projection (Satoda, 1995).

Plexin is a neuronal cell surface molecule that has been identified in Xenopus. cDNA cloning reveals that plexin has no homology to known neuronal cell surface molecules but possesses, in its extracellular segment, three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. The exogenous plexin proteins expressed on the surfaces of L cells by cDNA transfection mediate cell adhesion via a homophilic binding mechanism, under the presence of calcium ions. Plexin is expressed in the receptors and neurons of particular sensory systems. These findings indicate that plexin is a novel calcium-dependent cell adhesion molecule and suggest its involvement in specific neuronal cell interaction and/or contact (Ohta, 1995).

By screening E17.5 mouse brain cDNA libraries, two cDNAs encoding new plexin-like proteins were isolated. Sequencing revealed that these two proteins are type 1 membrane proteins that show over 60% identity to mouse plexin 1 at the amino acid level. Moreover, putative extracellular segments of these two proteins have three repeats of a cysteine-rich domain that is a common motif for plexin proteins. Thus, these two proteins have been named mouse plexin 2 and mouse plexin 3. Mouse plexin 3 cDNA clones in which a part of the protein-coding region had been deleted were obtained. Also, Northern blot analysis shows molecular heterogeneity in mouse plexin 2 mRNAs. These findings indicate that in the mouse, plexins comprise a molecular family (the plexin family) (Kameyama, 1996).

The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor has indicated that it is a novel member of the plexin family and has identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on human monocytes, and induced cytokine production from them as well. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs (Comeau, 1998).

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Plexins encode large transmembrane proteins whose cysteine-rich extracellular domains share regions of homology with the scatter factor receptors (encoded by the Met gene family). The extracellular domains of plexins also contain ~500 amino acid semaphorin domains. However, the highly conserved cytoplasmic moieties of plexins (~600 amino acids), have no homology with the Met tyrosine kinase domain nor with any other known protein. Met-like receptors and their ligands, the scatter factors, mediate a complex biological program including dissociation of cell-cell contacts, motility, and invasion. During embryogenesis, scatter factor-1 and Met promote the dissociation of cell layers in the somites and drive the migration of myogenic cells to their appropriate location. Met and scatter factor-1 are also involved in controlling neurite outgrowth and axonal guidance. The human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with an unidentified tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. It is concluded that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and that they trigger a novel signal transduction pathway controlling cell repulsion (Tamagnone, 1999).

Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance. Using yeast two-hybrid screening and in vitro interaction assays, it has been shown that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induces similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. The findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration (Driessens, 2001).

Many previously identified Rac targets contain a distinctive Rac binding site, the CRIB motif, but sequence analysis does not reveal any obvious CRIB-like sequence in plexin-B1. To identify the region of plexin-B1 that contains the Rac interaction site, a series of truncations were expressed as GST fusion proteins in E. coli. These were used in a dot blot assay. Rac interacts with a region encompassing 180 residues (amino acids 1724-1903) of the receptor (Driessens, 2001).

Plexin-B1 is a member of a large family of transmembrane proteins, and based on sequence alignments, four classes of plexins (A, B, C, and D) have been described. To test whether Rac could interact directly with other members of the family, cDNAs were obtained for human plexin-A2 (kiaa0463), plexin-B2 (kiaa0315), and plexin-D1 (kiaa0620). A region corresponding to amino acids 1724-1903 of plexin-B1 was cloned into the pGEX vector; GST fusion proteins were analyzed in the dot blot assay, but under these conditions only plexin-B1 was found to interact (Driessens, 2001).

In Drosophila, two plexins have been identified: Drosophila plexin-A and Drosophila plexin-B. Recombinant Drosophila plexin-B protein (C-terminal 435 amino acids, similar to plexin-B1 two-hybrid clone) interacts strongly with in vitro translated Drosophila L61Rac1 and weakly with wild-type Drosophila Rac1 in a pull-down experiment. Drosophila plexin-A does not interact with Drosophila Rac1 under the same conditions. A Drosophila plexin-B fragment corresponding to amino acids 1724-1903 of human plexin-B1 interacts similarly with Drosophila Rac1, as does a shorter, 149 amino acid region. Partial binding was observed with a 54 amino acid domain (Driessens, 2001).

Two blocks of sequence similarity, of approximately 320 and 150 amino acids each, have been identified in plexin cytoplasmic domains. These two blocks of sequence similarity are separated by a variable linker. This linker region is most divergent between the plexin subfamilies. The minimal Rac binding region in Drosophila plexin-B consists of the last 149 amino acids of the first conserved block but does not contain the linker region. Alignment of this 149 amino acid region of Drosophila plexin-B with other human plexins reveals a sequence highly conserved among all plexin subfamilies (Driessens, 2001).

A mechanism is proposed for plexin-B signaling to the actin cytoskeleton. In this mechanism, clustering of B plexins induces a Rac-dependent activation of Rho. These results provide a framework for the further exploration of the complex mechanisms by which plexins affect the actin cytoskeleton in different cell types, including neurons (Driessens, 2001).

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, these techniques have been used to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery (Brown, 2001).

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, evidence has been provided that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo (Cheng, 2001).

To test directly for a plexin-A3 signaling function, a gain-of-function approach was taken, asking whether the plexin-A3 cytoplasmic domain could mediate a repulsive response in the context of a chimeric receptor in which the extracellular domain of plexin-A3 is replaced by that of Met, a receptor for hepatocyte growth factor (HGF). This Met-plexin-A3 chimera was then introduced into embryonic Xenopus spinal neurons, which are repelled by Sema3A. Wild-type neurons do not respond to HGF but are attracted to HGF when wild-type Met is introduced in these cells. In contrast, when the chimeric Met-plexin-A3 receptor is introduced into these neurons, HGF elicites a robust repulsive response. This result provides evidence that plexin-A3 can function directly in signaling repulsion (Cheng, 2001).

Tracing studies have shown that developing hippocampal afferent axons invade their appropriate domains and layers in a highly specific fashion. Such stereotyped growth suggests the involvement of short-range cues providing layer-specific targeting information. Studies of reeler mutant mice deficient in reelin implicate a chemorepellent associated with Cajal-Retzius cells that inhibits commissural axon outgrowth into stratum lacunosum moleculare. Moreover, it has been suggested that Sema3F may play an important role in lamina-specific projections of hippocampal afferents. Plexin-A3 mutant commissural axons project to inappropriate laminae within the hippocampus, supporting the idea that the loss of plexin-A3 causes a reduction or loss in response to this Sema3F-based cue. Additionally, the observed switch in laminar termination zones also suggests the unmasking of an attractive cue within SLM. Laminar termination defects were not observed in spinal cord or cerebellum in plexin-A3 mutant mice, suggesting the importance of cues other than Sema3F in directing these terminations (Cheng, 2001).

A new member of the plexin-A subfamily has been identified in mice, plexin-A4: it was expressed in the developing nervous system with a pattern different from that of other members of the plexin-A subfamily (plexin-A1, plexin-A2 and plexin-A3). COS-7 cells coexpressing plexin-A4 with neuropilin-1 were induced by Sema3A, a member of the class 3 semaphorin, to undergo cell contraction. Ectopic expression of plexin-A4 in mitral cells that are originally insensitive to Sema3A results in the collapse of growth cones in the presence of Sema3A. These results suggest that plexin-A4 plays a role in the propagation of Sema3A activities. The strong expression of plexin-A4 in hippocampal neurons suggests the importance of plexin-A4 in hypocampus axon guidance (Suto, 2003).

Semaphorins, originally identified as axon guidance factors in the nervous system, play integral roles in organogenesis. A critical involvement is demonstrated for Sema6D in cardiac morphogenesis. Ectopic expression of Sema6D or RNA interference against Sema6D induces expansion or narrowing of the ventricular chamber, respectively, during chick embryonic development. Sema6D also exerts region-specific activities on cardiac explants, a migration-promoting activity on outgrowing cells from the conotruncal segment, and a migration-inhibitory activity on those from the ventricle. Plexin-A1 mediates these activities as the major Sema6D-binding receptor. Plexin-A1 forms a receptor complex with vascular endothelial growth factor receptor type 2 in the conotruncal segment or with Off-track in the ventricle segment; these complexes are responsible for the effects of Sema6D on the respective regions. Thus, the differential association of Plexin-A1 with additional receptor components entitles Sema6D to exert distinct biological activities at adjacent regions. This is crucial for complex cardiac morphogenesis (Toyofuku, 2004).

Plexin-A1 may form receptor complexes specific for Sema6D containing distinct components to exert opposite effects on different regions of the cardiac tube. The Drosophila Plexin-A receptor associates with OTK, a receptor tyrosine kinase-like transmembrane protein. This complex transduces Sema1a signals. The expression of various receptor kinases and NP1 was examined in outgrowing cells from cardiac explants. Among analyzed molecules, four molecules exhibited unique expression patterns. OTK and Neuropilin-1 (NP1) mRNAs are expressed predominantly in the outgrowing cells from the ventricle. In contrast, VEGFR1 and VEGFR2 mRNAs are strongly expressed in cells of the conotruncal segment. NP1 is known to form the functional Sema3A receptor complex with Plexin-A1. However, in spite of its abundant expression, the interaction of NP1 with Plexin-A1 could not be detected in cells from the ventricle, which is in good agreement with the fact that Sema3A does not show any effect on the ventricle explants. Since Drosophila OTK has been shown to interact with not only invertebrate Plexin-A, but also vertebrate Plexin-A3 and Plexin-B3, the ability of OTK to interact with Plexin-A1 was analyzed. An association between OTK and Plexin-A1 could be demonstrated when these molecules were transfected into HEK293 cells. The functional involvement of vertebrate OTK in Sema6D signaling was examined by knocking down OTK expression by RNAi. Treatment with siRNA specific for chicken OTK (cOTK) significantly reduces the levels of cOTK mRNA without changing any other mRNA tested. RNAi against cOTK blocked Sema6D-induced inhibition of migration of outgrowing cells from ventricle explants, but did not influence the effect of Sema6D on cells from conotruncal segments, indicating that OTK is functionally coupled with Plexin-A1 in the ventricle region. The effect of RNAi against cOTK on cardiac tube formation was also examined. Treatment with siRNA specific for cOTK, which significantly reduces the expression of cOTK mRNA, results in the failure of bending and expansion of the ventricular region. This phenotype is similar to those of chick embryos treated with siRNA specific for cSema6D or cPlexin-A1. These results suggest that the morphological effect of Sema6D on the ventricular region is mediated through the Plexin-A1-OTK receptor complex (Toyofuku, 2004).

Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. This study examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. The plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, a model is proposed for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2 (Suto, 2007).

Semaphorins and their receptors, plexins, carry out important functions during development and disease. In contrast to the well-characterized plexin A family, however, very little is known about the functional relevance of B-type plexins in organogenesis, particularly outside the nervous system. This study demonstrates that plexin B1 and its ligand Sema4d are selectively expressed in epithelial and mesenchymal compartments during key steps in the genesis of some organs. This selective expression suggests a role in epithelial-mesenchymal interactions. Importantly, using the developing metanephros as a model system, it was observed that endogenously expressed and exogenously supplemented Sema4d inhibits branching morphogenesis during early stages of development of the ureteric collecting duct system. The results further suggest that the RhoA-ROCK pathway, which is activated downstream of plexin B1, mediates these inhibitory morphogenetic effects of Sema4d and suppresses branch-promoting signalling effectors of the plexin B1 signalling complex. Finally, mice that lack plexin B1 show early anomalies in kidney development in vivo. These results identify a novel function for plexin B1 as a negative regulator of branching morphogenesis during kidney development, and suggest that the Sema4d-plexin B1 ligand-receptor pair contributes to epithelial-mesenchymal interactions during organogenesis via modulation of RhoA signalling (Korostylev, 2008).

A midline switch of receptor processing regulates commissural axon guidance in vertebrates

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, a molecular pathway was uncovered controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). Evidence is provided that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. At the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. During floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. The FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets (Nawabi, 2010).

In Drosophila, responsiveness of precrossing commissural axons to Slit is silenced through coupling of the Slit receptor Robo to Commissureless, and sorting for proteasome degradation. In vertebrates, a spliced variant of one of the three Robo gene products, Robo-3.1, appears as a functional equivalent of Drosophila Commissureless, preventing through a yet-undetermined mechanism Robo1 and Robo2 from mediating responsiveness to Slits at the precrossing stage. The present study highlights a novel mechanism by which precrossing commissural responses are silenced, based on processing of guidance receptors (Nawabi, 2010).

An interesting aspect of this pathway is that it does not prevent ligand/receptor interaction as for the Robo/Slit pair in Drosophila and possibly in vertebrates, since Nrp2 sorting to the growth cone surface is not prevented. Rather, it precludes accumulation of full-length signaling moiety of the receptor complex specifically transducing Sema3B in these neurons. This mechanism is advantageous and well-suited to the Semaphorin signaling, as it enables the other receptor subunit, Nrp2, to engage in other complexes (Nawabi, 2010).

Calpains are calcium-dependent cysteine proteases, regulating various processes. For example, they play pivotal roles in cell motility and synaptic functions by cleaving components of adhesion complexes and neurotransmitter receptors. Their functions during neuronal development are less characterized, but calpains are present in neuronal growth cones, are activated by intracellular calcium transients to reduce growth cone motility, and also are found downstream from Semaphorin5B. Interestingly, calpains process rather than degrade proteins, and are seen as regulators of protein functions, modulating protein-protein interactions, phosphorylation state, distribution, and traffic. The current data indicate that calpain1 is active in commissural neurons and maintains the integral form of Plexin-A1 at very low levels at the precrossing stage by cleaving the Plexin-A1 extracellular domain, generating two fragments that can be detected in Western blot. This processing could take place before protein sorting to the cell membrane. Likewise, previous studies reported detection of calpain in the lumen of endoplasmic reticulum and golgi vesicles, thus being at an appropriate location for cleaving neosynthesized target proteins during their intracellular traffic. Alternatively, Plexin-A1 could be cleaved at the cell surface, since increasing evidence indicates that calpains are externalized and can be retained to the membrane surface through association with proteoglycans. Several of the results support an important role for calpain activity during precrossing commissural axon guidance. First, in the spinal cord sections, calpains were found active in spinal neurons and precrossing commissural axon segments. Second, inhibition of calpain activity in vivo resulted in strong defects of commissural axon behaviors before FP crossing: axons failing to enter the FP, turning before FP crossing, or even not reaching the FP. Interestingly, all of these defects were also induced by Plexin-A1 overexpression in the chick embryo, thus supporting that calpain-1 activity prevents Plexin-A1 expression at the precrossing stage. Whether this protease suppresses precrossing commissural responses to other midline repellents by processing guidance receptors other than Plexin-A1, such as Robo1/2 or Eph receptors, is an intriguing possibility that will be assessed in future studies (Nawabi, 2010).

The results showed that suppression of calpain activity in the FP is instrumental for the gain of commissural responsiveness to Sema3B. Several data support this conclusion. First, in unfixed spinal cord sections, calpain were not active in FP cells or in the crossing axon tract. Second, it was found that the FP tissue released signals that inhibit calpain activity in spinal tissue. Third, suppression of calpain activity could increase integral Plexin-A1 levels in acute dorsal tissues and cultured commissural neurons and induce responsiveness to Sema3B (Nawabi, 2010).

Commissural axons were found to acquire responsiveness to a variety of repellents, but whether this occurs through a pathway-dependent or pathway-independent mechanism remains unclear. In the Xenopus visual system, the temporal switch from attractive to repulsive behavior of retinal axons to Netrin1 does not depend on pathway experience. The current data do not support the view that such an experience-independent mechanism operates to confer responsiveness to Sema3B at the midline. First, dorsal spinal neurons isolated from early E11 to late E13.5 were equally unresponsive to exogenous Sema3B application. Second, in open book preparations, removing the FP was sufficient to abolish the repulsive behavior of commissural neurons to a focal source of Sema3B. Finally, neurons became sensitive to Sema3B upon exposure to FP^cm (control supernatant), independent from the developmental stage at which they were collected. The data thus support that signals emanating from intermediate target cells play pivotal roles in the switch of responsiveness (Nawabi, 2010).

Consistently, it was shown that the Ig superfamily cell adhesion molecule NrCAM is an active FP component, regulating the Plexin-A1 level and acquisition of responsiveness to Sema3B. High levels of NrCAM transcripts were detected in the FP, and the protein was present in the FP^cm. In the neuronal assay, soluble NrCAM mimicked the FP^cm, triggering responsiveness to Sema3B and increase of the PlexinA1 level. In contrast, neither Netrin-1 nor Shh could recapitulate the gain of responsiveness to Sema3B conferred by the FP^cm. Moreover, the biochemical or genetic depletion of NrCAM strongly altered the properties of the FP^cm. In vivo, strong decrease of the Plexin-A1 level in the FP of NrCAM-null embryos was found and significant amounts of axon tracts abnormally stalled in the FP. NrCAM is expressed by various developing neuronal projections, and several previous studies implicated it in the regulation of axon navigation, as receptors or coreceptors for environmental guidance cues. Likewise, NrCAM interaction with Nrp2 was found to be required for axons to normally form the anterior commissure in the brain, and NrCAM expression is required by RGC axons to form proper patterns of ipsilateral/contralateral commissures in the visual system. Interestingly, NrCAM is highly expressed in specialized glial structures in the ventral midline -- not only in the spinal cord, but also in all upper floors of the CNS, but how NrCAM glial sources contribute to axon pathfinding remains unknown. The present work establishes a novel function for NrCAM as an intermediate target cue regulating the expression level of guidance receptors in the growth cones to control pathway choices at the ventral midline (Nawabi, 2010).

Neuropilins: co-receptors for semaphorins

Extending axons in the developing nervous system are guided to their targets through the coordinate actions of attractive and repulsive guidance cues. The semaphorin family of guidance cues comprises several members that can function as diffusible axonal chemorepellents. To begin to elucidate the mechanisms that mediate the repulsive actions of Collapsin-1/Semaphorin III/D (Sema III), a search in embryonic rat sensory neurons (using expression cloning) was carried out for Sema III-binding proteins. Sema III binds with high affinity to the transmembrane protein neuropilin; antibodies to neuropilin block the ability of Sema III to repel sensory axons and to induce the collapse of neuronal growth cones. Both the C domain and the semaphorin domain of Sema II can independently bind neuropilin. Neuropilin is an axonal protein present in the developing Xenopus nervous system. Neuropilin comprises in its extracellular domain two domains with similarity to the C1 and C2 domains of coagulation factors V and VIII; a MAM domain, and two CUB motifs (a CUB domain in the metalloproteinase Tolloid, a relative of BMP-1, is suggested to mediate an interaction with the BMP family member Decapentaplegic). These results provide evidence that neuropilin is a receptor or a component of a receptor complex that mediates the effects of Sema III on these axons (He, 1997).

Semaphorin III (Sema III) is a secreted protein that causes in vitro neuronal growth cone collapse and chemorepulsion of neurites, and in vivo is required for correct sensory afferent innervation and other aspects of development. However, the mechanism of Sema III function remains unknown. Neuropilin, a type I transmembrane protein implicated in aspects of neurodevelopment, is a Sema III receptor. Neuropilin-2, a related neuropilin family member, is described in this study. Both neuropilin and neuropilin-2 are expressed in overlapping, yet distinct, populations of neurons in the rat embryonic nervous system (Kolodkin, 1997).

Neuropilin is a neuronal cell surface protein that has been shown to function as a receptor for a secreted protein, semaphorin III/D, which can induce neuronal growth cone collapse and repulsion of neurites in vitro. Neuropilin is a type I membrane protein that is highly conserved among vertebrates, can mediate cell adhesion by a heterophilic molecular interaction, and can promote neurite outgrowth in vitro. The roles of neuropilin in vivo, however, are unknown. Neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. The trajectory of each cranial nerve is severely disorganized in the neuropilin mutant embryos. The ophthalmic nerve is defasciculated, and overshoots far beyond the growing front of the normal nerve. The distal parts of the maxillary and mandibular nerves are also difasciculated in mutants and spread into almost all parts of the maxillae and mandibula, respectively. The distal parts of the facial nerve, glossopharyngeal and vagus nerves also expand beyond their normal extentions. Spinal nerve fibers at the trunk level show abnormal trajectory and projection in Neuropilin mutants, and limb innervation by the fourth to eighth cervical spinal nerves is abnormal. Neuropilin-deprived dorsal root ganglion neurons are protected from growth cone collapse elicited by semaphorin III/D. These results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in the guidance of PNS efferents (Kitsukawa, 1997).

Neuropilin 1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, it has been demonstrated that neuropilins are sufficient to determine the functional specificity of semaphorin reponsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity toward NP-1 receptors, whereas they act as agonists at receptors containing NP-2 (Takahashi, 1998).

The collapsin and semaphorin family of extracellular proteins contributes to axonal path finding by repulsing axons and collapsing growth cones. To explore the mechanism of collapsin-1 action, a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4) was expressed. This protein retains biological activity as a DRG growth cone collapsing agent and saturably binds to DRG neurons with low nanomolar affinity. Specific CAP-4 binding sites are present on DRG neurons, sympathetic neurons, and motoneurons, but not on retinal, cortical, or brainstem neurons. Outside the nervous system, high levels of CAP-4 binding sites are present in the mesenchyme surrounding major blood vessels and developing bone and in lung. These sites provide a substrate for the collapsin-1-dependent patterning of non-neuronal tissues perturbed in sema III (-/-) mice. The staining patterns for mouse semaphorin D/III and chick collapsin-1 fusion proteins are indistinguishable from one another but quite separate from those for semaphorin B and M-semaphorin F fusion proteins. These data imply that there exists a family of high-affinity semaphorin binding sites similar in complexity to the semaphorin ligand family (Takahashi, 1997).

Neuropilin (neuropilin-1) was recently identified as a receptor for Collapsin-1/Semaphorin III/D (Sema III). A related protein has been identified, neuropilin-2, whose mRNA is expressed by developing neurons in a pattern largely, though not completely, nonoverlapping with that of neuropilin-1. Unlike neuropilin-1, which binds with high affinity to the three structurally related semaphorins (Sema III, Sema E, and Sema IV), neuropilin-2 shows high affinity binding only to Sema E and Sema IV, not Sema III. These results identify neuropilins as a family of receptors (or components of receptors) for at least one semaphorin subfamily. They also suggest that the specificity of action of different members of this subfamily may be determined by the complement of neuropilins expressed by responsive cells (Chen, 1997).

Neuropilin-1 and neuropilin-2 show specificity in binding to different class III semaphorins, including Sema III, Sema E, and Sema IV, suggesting that the specificity of action of these semaphorins is dictated by the complement of neuropilins expressed by responsive neurons. In support of this, sympathetic axons have been shown to coexpress neuropilin-1 and -2; their responses to Sema III, Sema E, and Sema IV are affected in predicted ways by antibodies to neuropilin-1, and neuropilin-1 and -2 can form homo- and hetero-oligomers through an interaction involving (at least partly) the neuropilin MAM (meprin, A5, mu) domain. These results support the idea that in sympathetic axons, the Sema III signal is mediated predominantly by neuropilin-1 oligomers; the Sema IV signal by neuropilin-2 oligomers, and the Sema E signal by neuropilin-1 and -2, either as homo- or hetero-oligomers (Chen, 1998).

Collapsin-1, a member of the semaphorin family, activates receptors on specific growth cones, thereby inhibiting their motility. Neuropilin, a previously cloned transmembrane protein, has recently been identified as a candidate receptor for collapsin-1. The cloning of chick collapsin-3 and -5 has been completed, and collapsin-1, -2, -3, and -5 are known to bind to overlapping but distinct axon tracts. In situ, there are inferred to be distinct receptors with different affinities for collapsin-1, -2, -3, and -5. In contrast, these four collapsins all bind recombinant neuropilin with similar affinities. Strong binding to neuropilin is mediated by the carboxy third of the collapsins, while the semaphorin domain confers collapsins' unique binding patterns in situ. It is proposed that neuropilin is a common component of a semaphorin receptor complex, and that additional differentially expressed receptor components interact with the semaphorin domains to confer binding specificity (Feiner, 1997).

To explore a role for chemorepulsive axon guidance mechanisms in the regeneration of primary olfactory axons, the expression of the chemorepellent semaphorin III (sema III), its receptor neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) were examined during regeneration of the olfactory system. In the intact olfactory system, neuropilin-1 and CRMP-2 mRNA expression define a distinct population of olfactory receptor neurons, corresponding to immature (B-50/GAP-43-positive) neurons, and a subset of mature (olfactory marker protein-positive) neurons, located in the lower half of the olfactory epithelium. Sema III mRNA is expressed in pial sheet cells and in second-order olfactory neurons that are the target cells of neuropilin-1-positive primary olfactory axons. These data suggest that in the intact olfactory bulb, sema III creates a molecular barrier, which helps restrict ingrowing olfactory axons to the nerve and glomerular layers of the bulb. Both axotomy of the primary olfactory nerve and bulbectomy induce the formation of new olfactory receptor neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema III mRNA is transiently induced in cells at the site of the lesion. These cells align regenerating bundles of olfactory axons. In contrast to the transient appearance of sema III-positive cells at the lesion site after axotomy, sema III-positive cells increase progressively after bulbectomy, apparently preventing regenerating neuropilin-1-positive nerve bundles from growing deeper into the lesion area. The presence of sema III in scar tissue and the concomitant expression of its receptor neuropilin-1 on regenerating olfactory axons suggests that semaphorin-mediated chemorepulsive signal transduction may contribute to the regenerative failure of these axons after bulbectomy (Pasterkamp, 1998).

The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic and motor axons. CA1, CA3 and dentate gyrus axons from E15-E17 mouse embryo explants are selectively repelled by entorhinal cortex and neocortex. The secreted semaphorins Sema III and Sema IV and their receptors Neuropilin-1 and -2 are expressed in the hippocampal formation during appropriate stages. Sema III and Sema IV strongly repel CA1, CA3 and dentate gyrus axons; entorhinal axons are only repelled by Sema III. An antibody against Neuropilin-1 blocks the repulsive action of Sema III and the entorhinal cortex, but has no effect on Sema IV-induced repulsion. Thus, chemorepulsion plays a role in axon guidance in the hippocampus; secreted semaphorins are likely to be responsible for this action, and the same axons can be repelled by two distinct semaphorins via two different receptors (Chedotal, 1998).

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). SemD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus- (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, it has been shown that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1: the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, mu) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein, which in turn transduces a semD signal into axon repulsion (Nakamura, 1998).

Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Neuropilin-2 is a receptor for the secreted semaphorin Sema IV and acts selectively to mediate repulsive guidance events in discrete populations of neurons. neuropilin-2 and semaIV are expressed in strikingly complementary patterns during neurodevelopment. The extracellular complement-binding (CUB) and coagulation factor domains of neuropilin-2 confer specificity to the Sema IV repulsive response, and these domains of neuropilin-1 are necessary and sufficient for binding of the Sema III semaphorin (sema) domain. The coagulation factor domains alone are necessary and sufficient for binding of the Sema III immunoglobulin- (Ig-) basic domain and the unrelated ligand, vascular endothelial growth factor (VEGF). Lastly, neuropilin-1 can homomultimerize and form heteromultimers with neuropilin-2. These results provide insight into how interactions between neuropilins and secreted semaphorins function to coordinate repulsive axon guidance during neurodevelopment (Giger, 1998).

The olfactory system provides an example of the complex expression pattern of Sema IV and Neuropilin-2. In the developing olfactory system, SemaIV is expressed in regions apical to the VNR neurons of the vomeronasal organ (VNO). Equally strong expression of neuropilin-2 is observed in the V2R neurons of the basal VNO. The V2R neurons initially project basally into the accessory olfactory nerve, away from the region of SemaIV expression in the VNO, commensurate with the idea that Sema IV serves to direct the initial projections of these neurons. The accessory olfactory bulb (OB), unlike the main OB, does not express high levels of SemaIV, and this lack of semaIV expression may serve to help segregate VNO projections to the accessory OB from olfactory epithelium (OE) projections to the main olfactory bulb. Neuropilin-2 is strongly expressed by both VNO neurons and their target region in the accessory OB. It is possible that homophilic Neuropilin-2 interactions may serve to help establish appropriate connectivity between the VNO and the accessory olfactory bulb and may even function to regulate fasciculation of accessory olfactory neurons. SemaIV is expressed in the main OE, with stronger expression observed in the basal OE, and since subsets of main olfactory receptor neurons (ORNs) also express Neuropilin-2, Sema IV may play a role as well in directing the initial outgrowth of ORNs to the olfactory nerve. In addition, SemaIV is expressed in the main OB in the periglomerular, mitral, and tufted cells and could prevent the main ORNs from overshooting their glomerular targets in the main OB (Giger, 1998).

Neuropilin-1 (Npn-1), a receptor for semaphorin III, mediates the guidance of growth cones on extending neurites. The molecular mechanism of Npn-1 signaling remains unclear. A yeast two-hybrid system has been used to isolate a protein that interacts with the cytoplasmic domain of Npn-1. This Npn-1-interacting protein (NIP) contains a central PSD-95/Dlg/ZO-1 (PDZ) domain and a C-terminal acyl carrier protein domain. The physiological interaction of Npn-1 and NIP is supported by co-immunoprecipitation of these two proteins in extracts from a heterologous expression system and from a native tissue. The C-terminal three amino acids of Npn-1 (S-E-A-COOH), which is conserved from Xenopus to human, is responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NIP. NIP as well as Npn-1 are broadly expressed in mice as assayed by Northern and Western analysis. Immunohistochemistry and in situ hybridization experiments reveal that NIP expression overlaps that of Npn-1. NIP has been independently cloned as RGS-GAIP-interacting protein (GIPC): it was identified by virtue of its interaction with the C terminus of RGS-GAIP and has been suggested to participate in clathrin-coated vesicular trafficking. It is suggested that NIP and GIPC may participate in regulation of Npn-1-mediated signaling as a molecular adapter that couples Npn-1 to membrane trafficking machinery in the dynamic axon growth cone (Cai, 1999).

While neuropilin-1 (NP-1) is necessary and sufficient for growth cone binding of Sema3A, NP-1 does not itself transmit a signal to the cytoplasmic domain of the growth cone. The two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1, form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes (Takahashi, 1999).

Several lines of evidence suggest that the signal transducer for the NP-1/Sema3A complex is a plexin: (1) NP-1 and Plex 1 form an immunoprecipitable complex; (2) the complex exhibits an enhanced affinity for Sema3A close to the Sema3A affinity seen in growth cones; (3) the affinity enhancement of the complex requires the signal-transducing MAM domain of NP-1 and is agonist selective; (4) the NP-1/Plex 1 complex is sufficient to mediate morphologic responses to Sema3A in nonneuronal cells; (5) the NP-1 structural requirements for Plex 1-mediated changes in nonneuronal cell morphology are the same as those for growth cone collapse; (6) the cytoplasmic domain of Plex 1 is essential for these Sema3A-induced morphologic changes; (7) plexin and NP-1 cocluster during Sema3A treatment of DRG growth cones, and (8) a dominant-negative form of Plex 1 dramatically reduces Sema3A-induced growth cone collapse. Taken together, the data strongly support the hypothesis that a plexin mediates the actions of Sema3A/NP-1 complexes (Takahashi, 1999).

The idea that the signal transducer for the NP-1/Sema3A complex is a plexin implies a unified scheme for the mechanism of semaphorin action. All semaphorin signals may be mediated via plexins. Soluble class 3 semaphorins utilize NPs as high-affinity binding intermediates in order to access a more general plexin transduction cascade. For class 1 and viral semaphorins, there is convincing evidence that plexins directly bind semaphorin ligands and mediate cellular effects. Other semaphorins may utilize plexin isoforms directly, or additional non-NP accessory components may exist. It is tempting to speculate that class 3 soluble semaphorins require NPs to enhance binding affinities, whereas membrane-associated semaphorins are confined to the plane of the lipid bilayer and require a lower binding affinity to achieve biological specificity, and this lower affinity is provided by plexins directly. This model also implies that plexins are bifunctional: they can stand alone as Sema-1 receptors and also serve as transducing subunits for Sema3A/NP-1 complexes (Takahashi, 1999).

The unified model of semaphorin signaling through plexins presented above emphasizes the paucity of information concerning how the intracellular domain of plexin might transduce a signal. It is noteworthy that the large intracellular domain is highly conserved across the plexin family but does not share strong sequence similarity to another protein, which might suggest an obvious hypothesis for signaling function. There is weak similarity of Plex 1 residues 1667-1825 with a group of R-ras GTPase-activating proteins (GAPs). However, a model for semaphorin signaling based on this similarity is not obvious. It is known that the NPs and some semaphorins self-associate. Therefore, their association with Plex 1 might create higher-order ligand-receptor complexes. Perhaps receptor aggregation activates a signaling function of the plexin intracellular domain. While Sema3A does not regulate the extent of coprecipitation of NP-1 with NP-1, or NP-1 with Plex 1, the immunohistologic data suggest that Sema3A induces higher-order receptor structures that in turn lead to the activation of signaling cascades and morphologic changes. In support of such a model, it has been found that Sema3A-induced NP-1/plexin clusters are associated with Rac1 aggregation, F-actin nucleation, membrane ruffles, and endocytosis (Takahashi, 1999).

Semaphorin 3A (Sema3A) binds to neuropilin-1 (NP1) and activates the transmembrane Plexin to transduce a repulsive axon guidance signal. Sema3 signals are transduced equally effectively by PlexinA1 or PlexinA2, but not by PlexinA3. Deletion analysis of the PlexinA1 ectodomain demonstrates that the sema domain prevents PlexinA1 activation in the basal state. Sema-deleted PlexinA1 is constitutively active, producing cell contraction, growth cone collapse, and inhibition of neurite outgrowth. The sema domain of PlexinA1 physically associates with the remainder of the PlexinA1 ectodomain and can reverse constitutive activation. Both the sema portion and the remainder of the ectodomain of PlexinA1 associate with NP1 in a Sema3A-independent fashion. Plexin A1 is autoinhibited by its sema domain, and Sema3A/NP1 releases this inhibition (Takahashi, 2001).

What is the role of the PlexA1 sema domain in the NP/PlexA1 complex? PlexA1sem associates with both NP1 and the remainder of PlexA1. Because coprecipitation is not modified by the presence of Sema3A, the PlexA1 sema domain must bind to NP1 at a site different from the Sema3A binding site of NP1. Indeed, coexpression of PlexA1sem with NP1 enhances Sema3A avidity for NP1. PlexA1Deltasem also associates with NP1 in a Sema3A-independent fashion and coexpression of PlexA1Deltasem increases Sema3A affinity for NP1. This implies that NP1 interacts with three partners at three distinct sites. PlexA1sem and the remainder of PlexA1 ectodomain cooperate to increase the Sema3A affinity for NP1. In the NP1/PlexA1 complex, PlexA1 sema domain interactions with both NP1 and the remainder of the PlexA1ectodomain may keep PlexA1 silent. Presumably, Sema3A binding to NP1 results in a dramatic conformational change in this complex. The simplest model is that this conformational change physically separates and functionally reduces PlexA1sem effects on the remainder of the PlexA1 ectodomain. In support of such a conformational dissociation of the two PlexA1 domains, excess PlexA1sem can reverse constitutive activation or Sema3A-induced PlexA1 activation. Although the two PlexA1 domains are functionally dissociated by Sema3A binding to NP-1, they remain bound in a NP1-dependent complex of altered conformation. The NP1 antagonists, Sema3C, Sema3B, and Sema3F, are expected to occupy the Sema3A site but not result in this conformational shift and the release of PlexA1sem from the remainder of the PlexA1 ectodomain (Takahashi, 2001).

In the developing nervous system axons navigate with great precision over large distances to reach their target areas. Chemorepulsive signals such as the semaphorins play an essential role in this process. The effects of one of these repulsive cues, semaphorin 3A (Sema3A), are mediated by the membrane protein neuropilin-1 (Npn-1). Neuropilin-1 is essential but not sufficient to form functional Sema3A receptors and indicates that additional components are required to transduce signals from the cell surface to the cytoskeleton. Members of the plexin family interact with the neuropilins and act as co-receptors for Sema3A. Neuropilin/plexin interaction restricts the binding specificity of neuropilin-1 and allows the receptor complex to discriminate between two different semaphorins. Deletion of the highly conserved cytoplasmic domain of Plexin-A1 or -A2 creates a dominant negative Sema3A receptor that renders sensory axons resistant to the repulsive effects of Sema3A when expressed in sensory ganglia. These data suggest that functional semaphorin receptors contain plexins as signal-transducing subunits and neuropilins as ligand-binding subunits (Rohm, 2000).

The class 3 Semaphorins Sema3A and Sema3F are potent axonal repellents that cause repulsion by binding Neuropilin-1 and Neuropilin-2, respectively. Plexins are implicated as signaling coreceptors for the Neuropilins, but the identity of the Plexins that transduce Sema3A and Sema3F responses in vivo is uncertain. This study shows that Plexin-A3 and -A4 are key determinants of these responses, through analysis of a Plexin-A3/Plexin-A4 double mutant mouse. Sensory and sympathetic neurons from the double mutant are insensitive to Sema3A and Sema3F in vitro, and defects in axonal projections in vivo correspond to those seen in Neuropilin-1 and -2 mutants. Interestingly, a differential requirement was found for these two Plexins: signaling via Neuropilin-1 is mediated principally by Plexin-A4, whereas signaling via Neuropilin-2 is mediated principally by Plexin-A3. Thus, Plexin-A3 and -A4 contribute to the specificity of axonal responses to class 3 Semaphorins (Yaron, 2005).

The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. This study shows that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. These data therefore reveal a 'gating' function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits (Chauvet, 2007).

PlexinA4 is necessary as a downstream target of Islet2 to mediate Slit signaling for promotion of sensory axon branching

Slit is a secreted protein known to repulse the growth cones of commissural neurons. By contrast, Slit also promotes elongation and branching of axons of sensory neurons. The reason why different neurons respond to Slit in different ways is largely unknown. Islet2 is a LIM/homeodomain-type transcription factor that specifically regulates elongation and branching of the peripheral axons of the primary sensory neurons in zebrafish embryos. PlexinA4, a transmembrane protein known to be a co-receptor for class III semaphorins, was shown to act downstream of Islet2 to promote branching of the peripheral axons of the primary sensory neurons. Intriguingly, repression of PlexinA4 function by injection of the antisense morpholino oligonucleotide specific to PlexinA4 or by overexpression of the dominant-negative variant of PlexinA4 counteracts the effects of overexpression of Slit2 to induce branching of the peripheral axons of the primary sensory neurons in zebrafish embryos, suggesting involvement of PlexinA4 in the Slit signaling cascades for promotion of axonal branching of the sensory neurons. Colocalized expression of Robo, a receptor for Slit2, and PlexinA4 is observed not only in the primary sensory neurons of zebrafish embryos but also in the dendrites of the pyramidal neurons of the cortex of the mammals, and may be important for promoting the branching of either axons or dendrites in response to Slit, as opposed to the growth cone collapse (Miyashita, 2004).

Signaling downstream of plexins

Plexins serve as receptors for repulsive axonal guidance molecules semaphorins. The cytoplasmic domain of the semaphorin 4D (Sema4D) receptor, Plexin-B1 has two separated Ras GTPase-activating protein (GAP)-homologous domains, C1 and C2. The Rho family small GTPase Rnd1 associates with Plexin-B1, and the Plexin-B1-Rnd1 complex stimulates GTPase activity of R-Ras, inducing growth cone collapse in hippocampal neurons in response to Sema4D. However, the molecular mechanisms by which Plexin-B1 exhibits the GAP activity remain unclear. This study examines critical roles of Rnd1 and Sema4D in Plexin-B1-stimulated R-Ras GAP activity and neurite remodeling. The N-terminal region of the cytoplasmic domain of Plexin-B1 containing the C1 domain interacts with the C-terminal region containing the C2 domain, and Rnd1 disrupts this interaction. In contrast, Sema4D induces clustering of Rnd1-bound Plexin-B1, in parallel with inactivation of R-Ras in cells. Antibody clustering of the recombinant cytoplasmic domain of Plexin-B1 in the presence of Rnd1 triggers the R-Ras GAP activity. Deletion of the extracellular domain of Plexin-B1 causes ligand-independent clustering of the receptor, rendering the receptor constitutively active in the presence of Rnd1, and induces contraction of COS-7 cells and inhibition of neurite outgrowth in hippocampal neurons. These results indicate that Rnd1 opens the two R-Ras GAP domains of Plexin-B1, and Sema4D-induced receptor clustering stimulates R-Ras GAP activity and neurite remodeling in hippocampal neurons (Oinuma, 2004).

The small GTPase Rac has been implicated in growth cone guidance mediated by semaphorins and their receptors. Plexin-B1, a receptor for Semaphorin4D (Sema4D), and p21-activated kinase (PAK) can compete for the interaction with active Rac and plexin-B1 can inhibit Rac-induced PAK activation. Expression of active Rac enhances the ability of plexin-B1 to interact with Sema4D. Active Rac stimulates the localization of plexin-B1 to the cell surface. The enhancement in Sema4D binding depends on the ability of Rac to bind plexin-B1. These observations support a model where signaling between Rac and plexin-B1 is bidirectional; Rac modulates plexin-B1 activity and plexin-B1 modulates Rac function (Vikis, 2002).

Sema4D enhances the interaction between plexin-B1 and active Rac. A model is proposed by which Sema4D binds the plexin-B1 receptor and stimulates the recruitment of Rac-GTP. Sequestration of Rac results in the inactivation of PAK and growth cone collapse/turning. This model conflicts with studies on the role of Rac downstream of the plexin-A1 receptor where dominant negative Rac inhibits collapse in response to Sema3A; this suggests that Rac activation is required for Sema3A-mediated growth cone collapse. Perhaps plexin-A and -B signal via different mechanisms since plexin-A does not interact with active Rac. However, in Drosophila, Rac functions downstream of plexA even though it does not interact with plexA. It is possible that a yet unidentified protein couples plexin-A with Rac (Vikis, 2002).

These results indicate that another consequence of the plexin-B1/Rac interaction is to modulate Sema4D ligand binding. This effectively classifies plexin-B1 as a downstream effector of Rac and is the first example of a small GTPase that directly regulates receptor function. An enhancement in the quantity of receptor at the cell membrane and minor changes in affinity for ligand contribute to this enhancement. Whether this is a result of enhanced recruitment to the cell surface and/or inhibition of receptor endocytosis is presently unclear. RhoA does not interact with plexin-B1 and does not stimulate Sema4D ligand binding, yet it has been reported to be activated by clustering of plexin-B1 receptor. In Drosophila, plexB interacts with Rho and stimulates its activity. It appears that humans and flies use different mechanisms for plexin-B stimulation of Rho activity (Vikis, 2002).

These data also suggest that endogenous Rac-GTP is necessary for the maintenance of plexin-B1 at the cell surface. This is based on the observation that dominant negative Rac (RacN17), which inhibits endogenous Rac activation, effectively inhibits the plexin-B1/Sema4D interaction. Furthermore, the Rac binding defective mutant plexin-B1-GGA is compromised in the interaction with Sema4D. This led the authors to postulate that factors that modulate Rac activation can enhance the sensitivity of the receptor/ligand interaction. It is possible that activation of a Rac-specific GEF and/or inactivation of a GAP may modulate the levels of plexin-B1 at the cell surface and its affinity for ligand. Hence, this suggests that engagement of plexin-B1 by Sema4D may be regulated by intracellular levels of Rac-GTP. What the biological consequence of this is remains unknown, however this may be a mechanism by which Rac activation by one axon guidance cue can modulate the responsiveness of the axon growth cone to another guidance cue, such as Sema4D. It is worth noting that whether this model operates in axon growth cone guidance requires further analysis in neurons. Under physiological conditions the axon growth cone is exposed to multiple guidance cues. Therefore, Rac may act as a mediator for cross-talk between different axon guidance cues. Furthermore, the data suggest that signaling between plexin-B1 and Rac is bidirectional. Ligand-gated plexin-B1 can sequester Rac from activating other downstream effectors whereas active Rac can enhance the activity of plexin-B1 (Vikis, 2002).

Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. Plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG (leukemia-associated Rho GEF). The molecular determinants of the interaction between plexin-B1 and PDZ-RhoGEF or LARG were analyzed. PDZ-RhoGEF mutants lacking the RGS domain or the DH/PH domain are still capable of interacting with plexin-B1 as effectively as wild-type, full-length PDZ-RhoGEF. In contrast, the PDZ-RhoGEF mutant lacking the PDZ domain does not interact with plexin-B1, indicating that the PDZ domain of PDZ-RhoGEF is required for its interaction with plexin-B1. Activation of plexin-B1 by semaphorin 4D regulates combined PDZ-RhoGEF and LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. This study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events that underlie various plexin-mediated cellular phenomena including axonal growth cone collapse (Swiercz, 2002).

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. This study shows that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration (Perrot, 2002).

Semaphorins are axon guidance molecules that signal through the plexin family of receptors. Semaphorins also play a role in other processes such as immune regulation and tumorigenesis. However, the molecular signaling mechanisms downstream of plexin receptors have not been elucidated. Semaphorin 4D is the ligand for the plexin-B1 receptor and stimulation of the plexin-B1 receptor activates the small GTPase RhoA. Using the intracellular domain of plexin-B1 as an affinity ligand, two Rho-specific guanine nucleotide exchange factors, leukemia-associated Rho GEF (LARG; GEF, guanine nucleotide exchange factors) and PSD-95/Dlg/ZO-1 homology (PDZ)-RhoGEF, were isolated from mouse brain as plexin-B1-specific interacting proteins. LARG and PDZ-RhoGEF contain several functional domains, including a PDZ domain. Biochemical characterizations showed that the PDZ domain of LARG is directly involved in the interaction with the carboxy-terminal sequence of plexin-B1. Mutation of either the PDZ domain in LARG or the PDZ binding site in plexin-B1 eliminates the interaction. The interaction between plexin-B1 and LARG is specific for the PDZ domain of LARG and LARG does not interact with plexin-A1. A LARG-interaction defective mutant of the plexin-B1 receptor was created and was unable to stimulate RhoA activation. The data in this report suggest that LARG plays a critical role in plexin-B1 signaling to stimulate Rho activation and cytoskeletal reorganization (Aurandt, 2002).

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). This study characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppresses R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressea the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocka the growth cone collapse. The R-Ras GAP activity is necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and is also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induces dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. It is concluded that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse (Ito, 2006).

Plexins are receptors for axonal guidance molecules known as semaphorins. The semaphorin 4D (Sema4D) receptor, Plexin-B1, induces axonal growth cone collapse by functioning as an R-Ras GTPase activating protein (GAP). This study reports that Plexin-B1 shows GAP activity for M-Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M-Ras is upregulated during dendritic development. Knockdown of endogenous M-Ras - but not R-Ras - reduces dendritic outgrowth and branching, whereas overexpression of constitutively active M-Ras, M-Ras(Q71L), enhances dendritic outgrowth and branching. Sema4D suppresses M-Ras activity and reduces dendritic outgrowth and branching, but this reduction is blocked by M-Ras(Q71L). M-Ras(Q71L) stimulates extracellular signal-regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppresses ERK activity and down-regulation of ERK is required for a Sema4D-induced reduction of dendrite growth. Thus, it is conclude that Plexin-B1 is a dual functional GAP for R-Ras and M-Ras, remodelling axon and dendrite morphology, respectively (Saito, 2009).

PlexinD1 and Semaphorin signaling are required in endothelial cells for cardiovascular development

The identification of new signaling pathways critical for cardiac morphogenesis will contribute to understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. A related signaling pathway involves secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has been previously attributed to abnormalities of neural crest. This study shows that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis (Gitler, 2004).

The closely related phenotypes involving the heart outflow tract (OT) and aortic arch defects of sema3C, plexinD1, and Npn-1 knockout mice, coupled with biochemical data, strongly suggest that semaphorin signaling mediated by endothelial cells (ECs) expressing PlexinD1 and neuropilins compose a receptor-ligand paracrine signaling pathway that orchestrates septation of the OT and development of aortic arch artery derivatives. Congenital heart defects in humans frequently involve OT and aortic arch defects, and SEMA3C, PLEXIND1, and NPN-1 are candidate genes for congenital heart disease. The ability of neuropilin to bind distinct ligands (VEGF¹⁶⁵ and SEMA3 proteins), coupled with elegant genetic studies, has suggested that neuropilin facilitates VEGF signaling in endothelium and semaphorin signaling in non-ECs including cardiac neural crest. Endothelial-specific loss of Npn-1 has been interpreted in terms of loss of VEGF¹⁶⁵ signaling in endothelium. However, biochemical and genetic data identify a direct role for neuropilin-mediated semaphorin signaling in endothelium. This result demands reinterpretation of existing data concerning the role of neuropilin, VEGF, and semaphorin signaling in cardiovascular development and led to the proposal of a unifying model. It is suggested that neuropilin, in ECs, functions in both VEGF and semaphorin signaling and that both pathways are required for proper cardiac OT development. Either Npn-1 or Npn-2 is able to cooperate with PlexinD1 to bind Sema3C. This explains why specific inhibition of Npn-1-dependent semaphorin signaling does not result in OT defects unless the redundant Npn-2 is also inactivated, whereas inactivating either the ligand Sema3C or the coreceptor PlexinD1 is sufficient to produce OT defects (Gitler, 2004 and references therein).

Semaphorin-Plexin signaling guides patterning of the developing vasculature

Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. Proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and mutations have been identified in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis (Torres-Vazquez, 2004).

Semaphorin 4D/Plexin-B1 stimulates PTEN activity through R-Ras GTPase-activating protein activity, inducing growth cone collapse in hippocampal neurons

Plexins are receptors for axonal guidance molecules semaphorins. The semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). This study examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. It is also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 alpha activity. Thus, it is proposed that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse (Oinuma, 2010).

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. This study shows that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL, ON and OFF referring to observations separate channels encoding light increments (ON) and decrements (OFF) are spawned in the outer retina and relayed to different sublaminas of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes (Matsuoka, 2011).

Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex

Co-receptors add complexity to cell-cell signaling systems. The secreted semaphorin 3s (Sema3s) require a co-receptor, neuropilin (Nrp), to signal through plexin As (PlxnAs) in functions ranging from axon guidance to bone homeostasis, but the role of the co-receptor is obscure. This study presents the low-resolution crystal structure of a mouse semaphorin-plexin-Nrp complex alongside unliganded component structures. Dimeric semaphorin, two copies of plexin and two copies of Nrp are arranged as a dimer of heterotrimers. In each heterotrimer subcomplex, semaphorin contacts plexin, similar to in co-receptor-independent signaling complexes. The Nrp1s cross brace the assembly, bridging between sema domains of the Sema3A and PlxnA2 subunits from the two heterotrimers. Biophysical and cellular analyses confirm that this Nrp binding mode stabilizes a canonical, but weakened, Sema3-PlxnA interaction, adding co-receptor control over the mechanism by which receptor dimerization and/or oligomerization triggers signaling (Janssen, 2012).

Neuropilins were identified as receptors for class 3 semaphorins before plexins were found to be the signal transducing receptors for these as well as other classes of semaphorins. These initial and subsequent studies showed that the Sema3s interact directly with the Nrps, and that for Sema3s to trigger PlxnA signaling the plexin and Nrp must be associated as a holoreceptor. How does this holoreceptor complex differ from a direct semaphorin - plexin complex and in what way does Nrp mediate ligand - receptor signaling in this system? In combination these studies indicate that Nrp is needed to cement a weak, but canonical, interaction between Sema3s and PlxnAs. The generic architecture of the semaphorin-plexin interaction as established by studies on other family members is conserved. It has been shown previously that semaphorin dimers are needed for signaling and the results presented in this study reveal that the core mechanism of semaphorin mediated plexin dimerization remains central to Sema3 function (Janssen, 2012).

Some twenty different semaphorins in higher vertebrates carry out a plethora of roles and the majority of these functions utilize one (or more) of the nine members of the plexin family of cell surface receptors for signal transduction. It is therefore perhaps unsurprising that a growing number of reports show diverse semaphorin ligands signal through the same plexin receptor to trigger very different cellular effects. The challenges posed for signal switching and fidelity are most apparent in the variety of biological functions mediated by PlxnAs. The PlxnAs bind multiple members of the class 3 and class 6 semaphorins. The Sema3s are secreted molecules and can modulate long-range effects on cellular processes by gradient formation. The current results explain the central role of Nrp, and specifically the a1 domain, in Sema3-PlxnA signaling. It is proposed that the requirement for a1 as a cross-brace stabilizing the Sema3-PlxnA complex allows Nrp to gate signaling through the PlxnA receptors, underpinning switches between Sema3 and Sema6 function such as recently reported for the osteoprotective activities of Sema3A. Similar to other semaphorin-plexin combinations, Sema3 binding dimerizes and possibly clusters the plexin intracellular region leading to signaling. Whilst the role of Nrp as a co-receptor specifically stabilizing Sema3-PlxnA complexes is revealed in this study, the potential contribution of Nrp dimerization to clustering, and thus the properties of the signaling assembly, remains open (Janssen, 2012).

Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. Little is known about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. This study shows that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. A novel, tissue-autonomous mechanism of organ cohesion is proposed, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain (Ebert, 2014).

On and off retinal circuit assembly by divergent molecular mechanisms

Direction-selective responses to motion can be to the onset (On) or cessation (Off) of illumination. This study shows that the transmembrane protein semaphorin 6A and its receptor plexin A2 are critical for achieving radially symmetric arborization of On starburst amacrine cell (SAC) dendrites and normal SAC stratification in the mouse retina. Plexin A2 is expressed in both On and Off SACs; however, semaphorin 6A is expressed in On SACs. Specific On-Off bistratified direction-selective ganglion cells in semaphorin 6A^-/- mutants exhibit decreased tuning of On directional motion responses. These results correlate the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion, shedding light on the development of visual pathways that use the same cell types for divergent outputs (Sun, 2013).

This study demonstrates that Sema6A, a classical axon guidance cue, is a molecular determinant that distinguishes On from Off visual pathways. Sema6A, together with PlexA2, regulates SAC dendritic stratification, On SAC dendritic morphology, and functional assembly of retinal direction-selective circuitry. In the neonatal murine retina, repulsive Sema6A-PlexA2 signaling disentangles On and Off SAC dendritic processes, providing the anatomical organization critical for the emergence and separation of On and Off direction-selective circuitry. Despite the observation of SAC inner plexiform layer stratification defects in all Sema6A^−/− and PlexA2^−/− mutant retinas examined, there remain normally stratified SAC processes in these mutants such that most SAC dendrites are still confined to their normal ChAT+ sublaminae, suggesting that additional dendritic stratification mechanisms function in parallel to Sema6A-PlexA2 signaling to ensure proper SAC dendrite stratification. The in vitro observation that exogenous Sema6A protein repels SAC neurites expressing PlexA2 but not Sema6A (corresponding to Off SACs), but does not affect SAC neurites expressing both PlexA2 and Sema6A (corresponding to On SACs), suggests that Sema6A and PlexA2 use in trans repulsion to facilitate correct SAC stratification. The lack of a repulsive response to exogenous Sema6A by SACs that express both Sema6A and PlexA2 likely reflects the silencing of PlexA2 by ligand expressed in cis, as has been observed in murine sensory neurons that express both Sema6A and PlexA4 and do not respond to exogenous Sema6A in vitro. The data suggest that On SAC dendritic processes in vivo are not repelled by exogenous Sema6A, so defects in their laminar stratification in the inner plexiform layer may occur as a secondary consequence of Off SAC stratification defects or as a result of distinct Sema6A-PlexA2 signaling interactions (Sun, 2013).

The Sema3A receptor Plexin-A1 suppresses supernumerary axons through Rap1 GTPases

The highly conserved Rap1 GTPases (see Drosophila Rap1) perform essential functions during neuronal development. They are required for the polarity of neuronal progenitors and neurons as well as for neuronal migration in the embryonic brain. Neuronal polarization and axon formation depend on the precise temporal and spatial regulation of Rap1 activity by guanine nucleotide exchange factors (GEFs) and GTPases-activating proteins (GAPs). Several Rap1 GEFs have been identified that direct the formation of axons during cortical and hippocampal development in vivo and in cultured neurons. However little is known about the GAPs that limit the activity of Rap1 GTPases during neuronal development. This study investigated the function of Sema3A and Plexin-A1 as a regulator of Rap1 GTPases during the polarization of hippocampal neurons. Sema3A was shown to suppress axon formation when neurons are cultured on a patterned substrate. Plexin-A1 (see Drosophila Plexin) functions as the signal-transducing subunit of receptors for Sema3A and displays GAP activity for Rap1 GTPases. Sema3A and Plexin-A1 suppress the formation of supernumerary axons in cultured neurons, which depends on Rap1 GTPases (Wang, 2018).

Architecture of the Sema3A/PlexinA4/Neuropilin tripartite complex

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. This study presents the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex (Lu, 2021).

While interactions of semaphorin with the plexin receptor and neuropilin co-receptor have been extensively investigated before, the Cryo-EM structure here provide a near complete view of the 2:2:2 extracellular region complex of these three large multi-domain proteins. The overall architecture of the 2:2:2 complex, dictated by multiple relatively weak interfaces contributed by each component, arranges the two plexin molecules in a way that can promote their activation and signaling. The order in which the three proteins assemble into the 2:2:2 complex could vary. The heterodimerization of both semaphorin and plexin could substantially diversify the composition of the 2:2:2 complexes, which may gain addition structural features and generate different signaling outputs. The ring-shape of the 10-domain extracellular region of class A plexins is essential for this activation mechanism, as its curvature allows the two copies of the membrane proximal IPT6 domain to converge and thereby induce the formation of the active dimer of the intracellular region. The ring-shape has also been shown to mediate the formation of the ligand-independent dimer of class A plexins that prevents spontaneous activation in the absence of the ligand by keeping the intracellular region in the monomeric state (Lu, 2021).

Prior to ligand binding, class A plexins form the inhibitory dimer that prevents the formation of the intracellular active dimer. It is possible that neuropilin uses its a1 domain to bind plexin, disrupting the inhibitory dimer and priming plexin for activation. Neuropilin could bind the semaphorin dimer on its own, considering the strong interaction between the two. Ultimately, the semaphorin dimer induces the formation of the 2:2:2 complex, which in turn induces the intracellular active dimer of plexin for signaling. Whether and how the transmembrane region of plexin and neuropilin interact in the 2:2:2 complex is unclear (Lu, 2021).

The ring-shape of PlexinA4 appears quite rigid in both the apo-state and the 2:2:2 complex. However, three crystal structures of the full-length extracellular region of PlexinA1 display substantial variations in the ring-shape. Docking one of these PlexinA1 structures to the cryo-EM structure based on superimposition of the Sema domain of PlexinA1 and PlexinA4 shows that this conformation of PlexinA1 can form a 2:2:2 active complex similar to that of PlexinA4. However, the same modelling of the other two PlexinA1 crystal structures results in severe clashes between the two IPT6 domains in the complex, suggesting that these conformations are not compatible with the complex formation or activation of PlexinA1. These observations suggest that the conformational flexibility in the extracellular region of different plexin family members may play a role in regulating their activation. Along this line, the relatively short linker (10-15 residues) between the IPT6 and transmembrane region of class A plexin may allow the transmembrane region to sense the difference in the distance between the two copies of the IPT6 domains in the 2:2:2 semaphorin/plexin/neuropilin complexes, thereby finely tuning the formation of the intracellular active dimer of plexin. This type of regulation in other receptors such as RET, the EGF receptor and c-Kit, has been shown to lead to differences in strength or duration of signaling, and in some cases biased signaling where different downstream pathways are activated to ultimately drive qualitatively distinct biological outcomes. Similar regulation of signaling in plexin may allow closely related plexin family members to carry out different functions, despite their overlapping ligand-binding specificities (Lu, 2021).

The Cryo-EM structure presented in this study clarifies the roles for the five domains and the interdomain linkers of neuropilin in the formation of the semaphorin/plexin/neuropilin complex. Interestingly, in both neuropilin and semaphorin, long flexible linkers are required for serving as spacers for the formation of the 2:2:2 complex. Such long linkers in proteins are often neglected in structural and functional studies because they are unstructured and nonconserved in sequence. These analyses suggest that serving as spacers might be a common function of long linkers, especially in large multi-protein assemblies. Related to this point, the MAM domain in neuropilin is not directly involved in binding of semaphorin or plexin, but truncation of this domain abolished the ability of neuropilin in mediating semaphorin-induced neuronal growth collapse. Early co-immunoprecipitation experiments suggested that the MAM domain regulate signaling by mediating dimerization or oligomerization. However, more recent structural and biophysical analyses have provided strong evidence for lack of dimerization of the MAM domain. Consistent with these results, the current structure shows that the two MAM domains are placed on opposite sides of the 2:2:2 semaphorin-plexin-neuropilin complex, unlikely to interact with each other. However, the MAM domain in neuropilin is a part of the linker-MAM-linker spacer that is required for the proper formation of the 2:2:2 semaphorin/plexin/neuropilin complex on the cell surface, which at least in part accounts for the functional importance of this domain in neuropilin. A remaining question is whether the linker-MAM-linker region in neuropilin connects to the transmembrane region through the center or outside of the ring of the plexin extracellular region. This question also pertains to how the transmembrane regions of plexin and neuropilin are organized and whether they form specific interactions in the 2:2:2 semaphorin/plexin/neuropilin complex. A structure of the complex of full-length semaphorin, plexin, and neuropilin is required for addressing these interesting mechanistic questions (Lu, 2021).

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date revised: 25 May 2024

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