Semaphorin-1a acts in concert with the cell adhesion molecules fasciclin II and connectin to regulate axon fasciculation in Drosophila.

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. Here, by manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, we provide further evidence that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons.

Expression of semaphorins in developing and regenerating olfactory epithelium.

Semaphorins provide signals that guide growing axons to their appropriate destinations. The secreted semaphorin, Sema3A, mediates repulsive effects on axons from various neuronal populations in embryonic rats. The authors localized Sema3A mRNA expression in the primary olfactory pathway during development, in adult rats, and in adult rats that were subjected to a unilateral olfactory bulbectomy. Developing rats at ages from embryonic day 14 (E14) to E19 expressed Sema3A in the olfactory receptor neurons (ORNs) of the olfactory epithelium and in chondrogenic structures surrounding the nasal cavity. In vitro, ORN axons at E14 avoided substrate-bound Sema3A. Low levels of Sema3A expression persisted in the normal adult epithelium both in ORNs scattered throughout the epithelium and in small clusters. Three days after a unilateral olfactory bulbectomy, Sema3A transcript levels increased in regenerating neurons. High levels of Sema3A transcript were found at 1 week postbulbectomy, persisted for 2 weeks, and diminished by 3 weeks. Several other murine semaphorins (Sema4A, Sema4B, and Sema4C) were expressed differentially in the primary olfactory pathway both during development and regeneration. These findings suggest that Sema3A and perhaps other semaphorins play a role in directing ORNs out of the epithelium and to the olfactory bulb, their target structure, during both development and regeneration.

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo.

From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, we have characterized a novel secreted semaphorin in grasshopper, gSema 2a. Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. We demonstrate that Sema 2a acts as chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo.

Patterning of cortical efferent projections by semaphorin-neuropilin interactions.

Cortical neurons communicate with various cortical and subcortical targets by way of stereotyped axon projections through the white matter. Slice overlay experiments indicate that the initial growth of cortical axons toward the white matter is regulated by a diffusible chemorepulsive signal localized near the marginal zone. Semaphorin III is a major component of this diffusible signal, and cortical neurons transduce this signal by way of the neuropilin-1 receptor. These observations indicate that semaphorin-neuropilin interactions play a critical role in the initial patterning of projections in the developing cortex.

Neuropilin-2 is a receptor for semaphorin IV: insight into the structural basis of receptor function and specificity.

Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Here, we show that 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.

The transmembrane Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon guidance.

The semaphorins comprise a large family of conserved glycoproteins, several members of which have been shown to function in repulsive neuronal growth cone guidance. We show here that Drosophila Semaphorin I (Sema I), a transmembrane semaphorin expressed on embryonic motor and CNS axons, is required for correct guidance of motor axons and for the formation of CNS pathways. In mutant embryos lacking Sema I, motor axons stall and fail to defasciculate at specific choice points where normally they would project to their muscle targets. In addition, a specific CNS fascicle fails to form correctly in these embryos. Rescue and ectopic expression experiments show that Sema I is required in neurons to mediate axon guidance decisions. These studies further suggest that like secreted semaphorins, transmembrane semaphorins can function as repulsive guidance cues for specific axon guidance events during neurodevelopment.

Neuropilin is a semaphorin III receptor.

The semaphorin family contains a large number of phylogenetically conserved proteins and includes several members that have been shown to function in repulsive axon guidance. Semaphorin III (Sema III) is a secreted protein that in vitro causes neuronal growth cone collapse and chemorepulsion of neurites, and in vivo is required for correct sensory afferent innervation and other aspects of development. The mechanism of Sema III function, however, is unknown. Here, we report that neuropilin, a type I transmembrane protein implicated in aspects of neurodevelopment, is a Sema III receptor. We also describe the identification of neuropilin-2, a related neuropilin family member, and show that neuropilin and neuropilin-2 are expressed in overlapping, yet distinct, populations of neurons in the rat embryonic nervous system.

Growth cones and the cues that repel them.

Neuronal growth cones establish appropriate connections with their targets during development by responding to both positive and negative guidance cues. The importance of repulsive and inhibitory cues in pathfinding and target selection has now been firmly established at the cellular and molecular levels. Observations in vitro have demonstrated developmentally significant repulsive interactions among various neuronal populations, providing the basis for molecular and functional characterization of several families of molecules that can mediate these guidance events. Analysis of both the expression and function of these molecules in vivo suggests how they, together with positive guidance cues, participate in the dynamic process of growth-cone guidance during both development and axonal regeneration.

Functions of netrins and semaphorins in axon guidance.

Neuronal growth cones respond to both contact-mediated and chemotropic guidance cues; these cues can be either attractive or repulsive. This past year has seen further characterization of two gene families implicated in long-range chemoattraction and chemorepulsion: the netrins and the semaphorins. Analysis of invertebrate members of these gene families demonstrates in vivo how netrins play multiple roles in axonal guidance in Caenorhabditis elegans, how specific domains of the netrin molecule confer attractive and repulsive guidance cues, and how semaphorins can function to generate neuromuscular specificity.

Semaphorins: mediators of repulsive growth cone guidance.

During development, neuronal growth cones encounter a variety of guidance cues while mediating axon path finding, target recognition and synapse formation. It is clear that repulsive guidance mechanisms play an essential role in these processes. The semaphorin gene family, which is conserved from invertebrates to mammals, includes members that mediate repulsive guidance. Molecular and cellular analysis of this gene family is providing insight into how inhibitory cues function during neurodevelopment.

Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord.

Distinct classes of primary sensory neurons in dorsal root ganglia subserve different sensory modalities, terminate in different dorsoventral locations in the spinal cord, and display different neurotrophin response profiles. Large diameter muscle afferents that terminate in the ventral spinal cord are NT-3 responsive, whereas small diameter afferents subserving pain and temperature are NGF responsive and terminate in the dorsal spinal cord. Previous in vitro studies showed that the developing ventral spinal cord secretes a diffusible factor that inhibits the growth of sensory axons. Here we show that this factor repels NGF-responsive axons but has little effect on NT-3-responsive axons. We also provide evidence implicating semaphorin III/collapsin, a diffusible guidance molecule expressed by ventral spinal cord cells, in mediating this effect. These results suggest that semaphorin III functions to pattern sensory projections by selectively repelling axons that normally terminate dorsally.

Semaphorin II can function as a selective inhibitor of specific synaptic arborizations.

Previous studies showed that grasshopper semaphorin I, a transmembrane semaphorin, functions in vivo to steer a pair of growth cones, prevent defasciculation, and inhibit branching; and that chick collapsin, a secreted semaphorin, can function in vitro to cause growth cone collapse. Semaphorin II, a secreted semaphorin in Drosophila, is transiently expressed by a single large muscle during motoneuron outgrowth and synapse formation. To test the in vivo function of semaphorin II, we created transgenic Drosophila that generate ectopic semaphorin II expression by muscles that normally do not express it. The results show that semaphorin II can function in vivo as a selective target-derived signal that inhibits the formation of specific synaptic terminal arbors.