Semaphorin Signaling Research Articles

Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin

Eye movements depend on correct patterns of connectivity between cranial motor axons and the extraocular muscles. Despite the clinical importance of the ocular motor system, little is known of the molecular mechanisms underlying its development. We have recently shown that mutations in the Chimaerin-1 gene encoding the signaling protein α2-chimaerin (α2-chn) perturb axon guidance in the ocular motor system and lead to the human eye movement disorder, Duane retraction syndrome (DRS). The axon guidance cues that lie upstream of α2-chn are unknown; here we identify candidates to be the Semaphorins (Sema) 3A and 3C, acting via the PlexinA receptors. Sema3A/C are expressed in and around the developing extraocular muscles and cause growth cone collapse of oculomotor neurons in vitro. Furthermore, RNAi knockdown of α2-chn or PlexinAs in oculomotor neurons abrogates Sema3A/C-dependent growth cone collapse. In vivo knockdown of endogenous PlexinAs or α2-chn function results in stereotypical oculomotor axon guidance defects, which are reminiscent of DRS, whereas expression of α2-chn gain-of-function constructs can rescue PlexinA loss of function. These data suggest that α2-chn mediates Sema3-PlexinA repellent signaling. We further show that α2-chn is required for oculomotor neurons to respond to CXCL12 and hepatocyte growth factor (HGF), which are growth promoting and chemoattractant during oculomotor axon guidance. α2-chn is therefore a potential integrator of different types of guidance information to orchestrate ocular motor pathfinding. DRS phenotypes can result from incorrect regulation of this signaling pathway.

Getting neural circuits into shape with semaphorins

Semaphorins are key players in the control of neural circuit development. Recent studies have uncovered several exciting and novel aspects of neuronal semaphorin signalling in various cellular processes--including neuronal polarization, topographical mapping and axon sorting--that are crucial for the assembly of functional neuronal connections. This progress is important for further understanding the many neuronal and non-neuronal functions of semaphorins and for gaining insight into their emerging roles in the perturbed neural connectivity that is observed in some diseases. This Review discusses recent advances in semaphorin research, focusing on novel aspects of neuronal semaphorin receptor regulation and previously unexplored cellular functions of semaphorins in the nervous system.

Rare Copy Number Variations in Adults with Tetralogy of Fallot Implicate Novel Risk Gene Pathways

Structural genetic changes, especially copy number variants (CNVs), represent a major source of genetic variation contributing to human disease. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, but to date little is known about the role of CNVs in the etiology of TOF. Using high-resolution genome-wide microarrays and stringent calling methods, we investigated rare CNVs in a prospectively recruited cohort of 433 unrelated adults with TOF and/or pulmonary atresia at a single centre. We excluded those with recognized syndromes, including 22q11.2 deletion syndrome. We identified candidate genes for TOF based on converging evidence between rare CNVs that overlapped the same gene in unrelated individuals and from pathway analyses comparing rare CNVs in TOF cases to those in epidemiologic controls. Even after excluding the 53 (10.7%) subjects with 22q11.2 deletions, we found that adults with TOF had a greater burden of large rare genic CNVs compared to controls (8.82% vs. 4.33%, p = 0.0117). Six loci showed evidence for recurrence in TOF or related congenital heart disease, including typical 1q21.1 duplications in four (1.18%) of 340 Caucasian probands. The rare CNVs implicated novel candidate genes of interest for TOF, including PLXNA2, a gene involved in semaphorin signaling. Independent pathway analyses highlighted developmental processes as potential contributors to the pathogenesis of TOF. These results indicate that individually rare CNVs are collectively significant contributors to the genetic burden of TOF. Further, the data provide new evidence for dosage sensitive genes in PLXNA2-semaphorin signaling and related developmental processes in human cardiovascular development, consistent with previous animal models.

Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development

BackgroundCortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled.ResultsWe report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate.ConclusionsThis analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.

Calpain cleaves and activates the TRPC5 channel to participate in semaphorin 3A-induced neuronal growth cone collapse

The nonselective cation channel transient receptor potential canonical (TRPC)5 is found predominantly in the brain and has been proposed to regulate neuronal processes and growth cones. Here, we demonstrate that semaphorin 3A-mediated growth cone collapse is reduced in hippocampal neurons from TRPC5 null mice. This reduction is reproduced by inhibition of the calcium-sensitive protease calpain in wild-type neurons but not in TRPC5(-/-) neurons. We show that calpain-1 and calpain-2 cleave and functionally activate TRPC5. Mutation of a critical threonine at position 857 inhibits calpain-2 cleavage of the channel. Finally, we show that the truncated TRPC5 predicted to result from calpain cleavage is functionally active. These results indicate that semaphorin 3A initiates growth cone collapse via activation of calpain that in turn potentiates TRPC5 activity. Thus, TRPC5 acts downstream of semaphorin signaling to cause changes in neuronal growth cone morphology and nervous system development.

Abstract LB-404: Genomic analysis reveals roles for chromatin modification and axon guidance in pancreatic cancer

Abstract Pancreatic cancer (PC) is the fourth leading cause of cancer death with an overall 5-year survival rate of < 5%, a statistic that has not changed in almost 50 years. A deeper understanding of the underlying molecular pathophysiology is expected to advance the urgent need to develop effective therapeutic and early detection strategies for this disease. Genomic characterisation of Pancreatic Ductal Adenocarcinoma (PDAC), which accounts for over 90% of PC has to date relied on targeted PCR based exome sequencing of primary and metastatic lesions propagated as xenografts or cell lines. Here we use exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (Stage I and II) PDAC. Detailed analysis of 99 informative tumours identified 1984 non-silent mutations and 1628 significant CNV events, and defined 439 significantly mutated genes based on stringent Significant Mutated Gene and GISTIC analysis. Integration with functional data from in vitro shRNA and in vivo Sleeping Beauty-mediated somatic mutagenesis screens provided supportive evidence for 182 of these as candidate driver mutations. Pathway based analysis recapitulated clustering of mutations in core signalling pathways in PDAC, and identified multiple new components in each, particularly in DNA damage repair mechanisms (ATM, TOP2A, RPA1, BRCC3). We also identified frequent and diverse somatic aberrations in genes involved in chromatin modification (SWI/SNF complex members, SETD2, EPC1), and novel mechanisms such as axon guidance (Semaphorin, Slit, Netrin and Ephrin signalling), extending the number of core perturbed pathways in PDAC. Aberrant expression of axon guidance genes co-segregated with poor patient survival, and in animal models was associated with disease development and progression, further implicating perturbation of the axon guidance pathway as a novel mechanism important in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-404. doi:1538-7445.AM2012-LB-404

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. We now uncover a simple biochemical switch that controls Semaphorin/Plexin repulsive guidance. Plexins are Ras/Rap family GTPase activating proteins (GAPs) and we find 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 we find to be 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. Our results indicate that theseinteractions switch repulsion to adhesion and identify a point of convergence for multiple guidance molecules.

PlexinA4 distribution in the adult rat spinal cord and dorsal root ganglia

PlexinsA1–A4 participate in class 3 semaphorin signaling as co-receptors to neuropilin 1 and 2, PlexinA4 being the latest member of the PlexinA subfamily to be identified. Little is known about the cellular distribution of PlexinA4 in the spinal cord and dorsal root ganglion (DRG). Here, immunohistochemical studies using antibodies to PlexinA4 revealed immunolabeling in neurons in both dorsal and, to a greater extent, ventral horns of the spinal cord. Ventral horn PlexinA4 positive neurons exhibited morphology, size, and location consistent with both motor neurons and interneurons. Labeling was found in motor axons exiting through the ventral roots, and more widespread labeling was observed in ascending and descending white matter tracts. Within the DRG, immunostaining was observed in neuronal cell bodies as well as the central and peripheral processes of these cells. PlexinA4 is expressed in the peripheral nervous system where its expression is regulated upon nerve injury. This is the first detailed description of the cellular and subcellular distribution of PlexinA4 in the adult spinal cord and DRG, and it will set the basis for future studies on the potential role of PlexinA4 in regeneration and repair of the adult central and peripheral nervous system.

Intercellular Cross-Talk Among Bone Cells: New Factors and Pathways

Intercellular communication within the bone microenvironment is critical for the maintenance of normal bone structure. Osteoblast-lineage cells at all stages of differentiation, from pluripotent precursors to matrix-embedded osteocytes, produce regulatory factors that modulate the differentiation and activity of both bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoclasts can also release factors that feed back to regulate osteoblast activity. Intercellular cross-talk within the bone microenvironment is not restricted only to these bone cells. Other cells within the bone marrow microenvironment, including adipocytes, T cells, and macrophages, play key roles that influence the processes of bone formation and resorption. This review discusses recent work that provides new insights into some of these communication networks and the factors involved, including osteocytic production of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin, osteoblastic production of interleukin-33, osteoclast-derived Semaphorin 4D, ephrin signaling, and signals from T helper cells and resident osteal macrophages (osteomacs).

Semaphorin Signaling in Vertebrate Neural Circuit Assembly

Neural circuit formation requires the coordination of many complex developmental processes. First, neurons project axons over long distances to find their final targets and then establish appropriate connectivity essential for the formation of neuronal circuitry. Growth cones, the leading edges of axons, navigate by interacting with a variety of attractive and repulsive axon guidance cues along their trajectories and at final target regions. In addition to guidance of axons, neuronal polarization, neuronal migration, and dendrite development must be precisely regulated during development to establish proper neural circuitry. Semaphorins consist of a large protein family, which includes secreted and cell surface proteins, and they play important roles in many steps of neural circuit formation. The major semaphorin receptors are plexins and neuropilins, however other receptors and co-receptors also mediate signaling by semaphorins. Upon semaphorin binding to their receptors, downstream signaling molecules transduce this event within cells to mediate further events, including alteration of microtubule and actin cytoskeletal dynamics. Here, I review recent studies on semaphorin signaling in vertebrate neural circuit assembly, with the goal of highlighting how this diverse family of cues and receptors imparts exquisite specificity to neural complex connectivity.

Semaphorin and Eph Receptor Signaling Guide a Series of Cell Movements for Ventral Enclosure in C. elegans

In the last stage of the Caenorhabditis elegans body wall closure, an open pocket in the epidermis is closed by the migration of marginal epidermal P/pocket cells to the ventral midline. The cellular and molecular mechanisms of this closure remain unknown. Cells within the pocket align to form a bridge for migration of contralateral P cell pair P9/10 L,R (and neighboring P cells) to the midline. Bridge formation involves rearrangement of five sister pairs of PLX-2/plexin and VAB-1/Eph receptor expressing "plexin band" cells, of which three pairs form a scaffold for bridge assembly and two pairs form the bridge. Bridge formation requires VAB-1 kinase-dependent extension of presumptive bridge cells over scaffold cells toward the ventral midline. An unassembled vab-1 null mutant bridge obstructs P cell migration, which is largely overcome by plexin band expression of VAB-1 or VAB-1(delC) (a kinase deletion of VAB-1). VAB-1 also functions redundantly with MAB-20/semaphorin to prevent perdurant gaps between sister plexin band cells that block P cell migration. The Eph receptor mediates cellular extensions required for bridge formation, independently facilitates P cell migration to the midline, and functions redundantly with PLX-2/plexin to prevent gaps in the bridge used for P9/10 cell migration in body wall closure.

In Vivo Gene Expression Profiling of Retina Postintravitreal Injections of Dexamethasone and Triamcinolone at Clinically Relevant Time Points for Patient Care

To identify retinal genes and their relevant expression pathways affected by intravitreal injections of dexamethasone (Dex) and triamcinolone acetonide (TAA) in mice at clinically relevant time points for patient care. Differential gene expressions of over 34,000 well-characterized mouse genes, in the retinas of 6-week-old C57BL/6J mice, were analyzed after intravitreal steroid injections at 1 week and 1 month time points, using mouse genome microarrays. The data were analyzed using commercial microarray analysis software for biologically relevant changes in gene expression pathways. A common gene pathway, with differentially activated genes for both steroids and time points, was "Semaphorin Signaling in Neurons," a member of the "Axonal Guidance Signaling System." At 1 week postinjection a common theme was activation of genes expressed in retinal glial cells, tumor necrosis factor-α, and transforming growth factor-β signaling pathways and upregulation of stress response proteins (Serpina3n, Cebpd), as well as neuropeptide signaling somatostatin receptor (Sstr2). Unique for Dex was the upregulation of acute phase proteins (Gfap, Cp, Edn2) as well as Plexna2, a semaphorin signaling receptor, whereas EphrinB receptor ephexin 1 (Argef15) was downregulated. Folate signaling appears to be unique for TAA at 1 week (Folh1, Cubn), whereas aryl-hydrocarbon receptor signaling might be important for both steroids at 1 month postinjection. Understanding the molecular and genetic effects of intraocular steroid treatments is of clinical relevance. This in vivo study has elucidated several genes and pathways that are potentially altering the neuroprotective/neurodegenerative balance between glial and retinal ganglion cells during intravitreal steroid treatment.

Semaphorins in angiogenesis and tumor progression.

The semaphorins were initially described as axon guidance factors, but have recently been implicated in a variety of physiological and developmental functions, including regulation of immune response, angiogenesis, and migration of neural crest cells. The semaphorin family contains more than 30 genes divided into seven subfamilies, all of which are characterized by the presence of a sema domain. The semaphorins transduce their signals by binding to one of the nine receptors belonging to the plexin family, or, in the case of the class 3 semaphorins, by binding to one of the two neuropilin receptors. Additional receptors, which form complexes with these primary semaphorin receptors, are also frequently involved in semaphorin signaling. Recent evidence suggests that some semaphorins can act as antiangiogenic and/or antitumorigenic agents whereas other semaphorins promote tumor progression and/or angiogenesis. Furthermore, loss of endogenous inhibitory semaphorin expression or function on one hand, and overexpression of protumorigenic semaphorins on the other hand, is associated with the progression of some tumor types.

Matrix Metalloproteinase-8 Is Involved in Dermal Nerve Growth: Implications for Possible Application to Pruritus from In Vitro Models

Cutaneous nerve density is related to abnormal itch perception in dermatoses, such as atopic dermatitis and xerosis. However, the mechanisms underlying the elongation of dermal nerve fibers within the interstitial collagen (CoL) matrix are poorly understood. In this study, a culture system of rat dorsal root ganglion neurons consisting of type I CoL and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. Nerve fibers penetrating into type I CoL gel were observed in the presence of the NGF concentration gradient. Levels of matrix metalloproteinase-8 (MMP-8) mRNA and protein were increased in the cultured neurons and the conditioned medium, respectively. The nerve fiber penetration was dose dependently inhibited by MMP-8 blockers. Moreover, MMP-8 immunoreactivity was partially localized at growth cones in NGF-responsive nerve fibers. Semaphorin 3A stimulation also showed the opposite effects on these NGF-dependent events. Intriguingly, MMP-8 expression was upregulated by type I and III CoLs, which are substrates for this enzyme. These results suggested that MMP-8 is involved in sensory nerve growth within the interstitial CoL matrix through modulation by the axonal guidance molecules and/or extracellular matrix components. These findings provide insight into the development of pruritus involving skin nerve density.

A shift of the TOR adaptor from Rictor towards Raptor by semaphorin in C. elegans.

The target of rapamycin (TOR), a central regulator for cell growth and metabolism, resides in the two functionally distinct complexes TORC1 and TORC2, which are defined by their adaptors Raptor and Rictor, respectively. How the formation of the two TORCs is orchestrated remains unclear. Here we show the control of TOR partnering by semaphorin-plexin signalling in Caenorhabditis elegans. In semaphorin and plexin mutants, TOR-Raptor association decreases whereas TOR-Rictor association increases, concomitantly with TORC1 down- and TORC2 up-regulation. Epidermal defects in the mutants are suppressed by inhibiting TORC2 or reinforcing TORC1 signalling. Conversely, inhibition of TORC1 signalling phenocopies the mutants. Thus, our results indicate that TORC formation is a singularly important step in semaphorin signalling that culminates in diverse outcomes including TORC1-promoted messenger RNA translation and TORC2-regulated cytoskeletal remodelling.

Frequent deletion of 3p21.1 region carrying semaphorin 3G and aberrant expression of the genes participating in semaphorin signaling in the epithelioid type of malignant mesothelioma cells

Array-based comparative genomic hybridization analysis was performed on 21 malignant mesothelioma (MM) samples (16 primary cell cultures and 5 cell lines) and two reactive mesothelial hyperplasia (RM) primary cell cultures. The RM samples did not have any genomic losses or gains. In MM samples, deletions in 1p, 3p21, 4q, 9p21, 16p13 and 22q were detected frequently. We focused on 3p21 because this deletion was specific to the epithelioid type. Especially, a deletion in 3p21.1 region carrying seven genes including SEMA3G was found in 52% of MM samples (11 of 14 epithelioid samples). The allele loss of 3p21.1 might be a good marker for the epithelioid MM. A homozygous deletion in this region was detected in two MM primary cell cultures. A heterozygous deletion detected in nine samples contained the 3p21.1 region and 3p21.31 one carrying the candidate tumor suppressor genes such as semaphorin 3F (SEMA3F), SEMA3B and Ras association (RalGDS/AF-6) domain family member1 (RASSF1A). SEMA3B, 3F and 3G are class 3 semaphorins and inhibit growth by competing with vascular endothelial growth factor (VEGF) through binding to neuropilin. All MM samples downregulated the expression of more than one gene for SEMA3B, 3F and 3G when compared with Met5a, a normal pleura-derived cell line. Moreover, in 12 of 14 epithelioid MM samples the expression level of SEMA3A was lower than that in Met5a and the two RM samples. An augmented expression of VEGFA was detected in half of the MM samples. The expression ratio of VEGFA/SEMA3A was significantly higher in the epithelioid MMs than in Met5a, RMs and the non-epithelioid MMs. Our data suggest that the downregulated expression of SEMA3A and several SEMA3s results in a loss of inhibitory activities in tumor angiogenesis and tumor growth of VEGFA; therefore, it may play an important role on the pathogenesis of the epithelioid type of MM.

Semaphorin Signals on the Road of Endothelial Tip Cells

Blood vessels sprout toward avascular tissue in response to attractive proangiogenic factors. However, restricting signals are also required to coordinate the behavior of endothelial cells assembling the vascular network. Semaphorin cues are at the crossroad of this traffic, and they direct the behavior of endothelial tip cells leading the way.

Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

Tbx1, subpulmonary myocardium and conotruncal congenital heart defects

Conotruncal congenital heart defects, including defects in septation and alignment of the ventricular outlets, account for approximately a third of all congenital heart defects. Failure of the left ventricle to obtain an independent outlet results in incomplete separation of systemic and pulmonary circulation at birth. The embryonic outflow tract, a transient cylinder of myocardium connecting the embryonic ventricles to the aortic sac, plays a critical role in this process during normal development. The outflow tract (OFT) is derived from a population of cardiac progenitor cells called the second heart field that contributes to the arterial pole of the heart tube during cardiac looping. During septation, the OFT is remodeled to form the base of the ascending aorta and pulmonary trunk. Tbx1, the major candidate gene for DiGeorge syndrome, is a critical transcriptional regulator of second heart field development. DiGeorge syndrome patients are haploinsufficient for Tbx1 and present a spectrum of conotruncal anomalies including tetralogy of Fallot, pulmonary atresia, and common arterial trunk. In this review, we focus on the role of Tbx1 in the regulation of second heart field deployment and, in particular, in the development of a specific population of myocardial cells at the base of the pulmonary trunk. Recent data characterizing additional properties and regulators of development of this region of the heart, including the retinoic acid, hedgehog, and semaphorin signaling pathways, are discussed. These findings identify future subpulmonary myocardium as the clinically relevant component of the second heart field and provide new mechanistic insight into a spectrum of common conotruncal congenital heart defects.

Robo1 Regulates Semaphorin Signaling to Guide the Migration of Cortical Interneurons through the Ventral Forebrain

Cortical interneurons, generated predominantly in the medial ganglionic eminence, migrate around and avoid the developing striatum in the subpallium en route to the cortex. This is attributable to the chemorepulsive cues of class 3 semaphorins expressed in the striatal mantle and acting through neuropilin (Nrp1 and Nrp2) receptors expressed in these cells. Cortical interneurons also express Robo receptors, and we show here that in mice lacking Robo1, but not Robo2, these cells migrate aberrantly through the striatum. In vitro experiments demonstrated that interneurons lacking Robo1 function are significantly less responsive to the effects of semaphorins. Failure to respond to semaphorin appears to be attributable to a reduction in Nrp1 and PlexinA1 receptors within these cells. Biochemical studies further demonstrated that Robo1 binds directly to Nrp1, but not to semaphorins, and this interaction is mediated by a region contained within its first two Ig domains. Thus, we show for the first time that Robo1 interacts with Nrp1 to modulate semaphorin signaling in the developing forebrain and direct the migration of interneurons through the subpallium and into the cortex.