Robo Receptors Research Articles

Dystroglycan Organizes Axon Guidance Cue Localization and Axonal Pathfinding

Precise patterning of axon guidance cue distribution is critical for nervous system development. Using a murine forward genetic screen for novel determinants of axon guidance, we identified B3gnt1 and ISPD as required for the glycosylation of dystroglycan invivo. Analysis of B3gnt1, ISPD, and dystroglycan mutant mice revealed a critical role for glycosylated dystroglycan in the development of several longitudinal axon tracts. Remarkably, the axonal guidance defects observed in B3gnt1, ISPD, and dystroglycan mutants resemble several of the axon guidance defects found in mice lacking the axon guidance cue Slit and its receptor Robo. This similarity is explained by our observations that dystroglycan binds directly to Slit and is required for proper Slit localization within the basement membrane and floor plate invivo. These findings establish a novel role for glycosylated dystroglycan as a key determinant of axon guidance cue distribution and function in the mammalian nervous system.

SLIT1 Mutation in Patients with Acquired Aplastic Anemia: Its Relevance in Immune Pathophysiology

Abstract 645 Background:Acquired aplastic anemia (AA) is a non-clonal hematopoietic disorder caused by the immune system attack on hematopoietic stem cells (HSCs) and has been thought to be rarely associated with intrinsic HSC defects. However, clonal hematopoiesis by HSCs with genetic alterations may not be uncommon in AA since clonal granulocyte populations showing the paroxysmal nocturnal hemoglobinuria (PNH) phenotype or chromosome 6p uniparental disomy (6pUPD) are detected in approximately 50% and 13% of AA patients, respectively (Katagiri T, et al, Blood, 2011). Abnormal HSCs other than those with the PIG-A mutation or 6pUPD may also contribute to hematopoiesis in AA patients due to immune escape or preferential activation of HSCs with genetic alterations in response to immune pressure in the bone marrow (BM). Objectives/methods:To identify novel genetic abnormalities in patients with AA, we carried out whole exome sequencing of peripheral blood leukocytes from 96 AA patients using a next generation sequencer. Among 40 abnormalities found in more than one patient, we selected abnormalities in genes potentially related to HSC regulation and examined the relevance of the genetic alterations to BM failure. Results:Missense mutations of SLIT1, a family of secreted glycoproteins that bind members of the Robo receptor family and regulate axon guidance, were detected in 15 of 96 patients and confirmed by Sanger sequencing in 3 patients who had been in remission for 1–3 years after immunosuppressive therapy at the time of blood sampling. Since previous studies showed that the Slit-Robo pathway negatively regulates proliferation of murine HSCs as well as a human cancer cell line in an autocrine manner (Prasad A et al., J Biol Chem; 283: 26624. 2008) and the break point of chromosome 10 in a patient with immune-mediated AA possessing t(1:10) was located within the SLIT1 gene, we studied SLIT1 and Robo receptor expression in BM mononuclear cells (BMMCs) obtained from healthy volunteers and patients with AA, and in myeloid leukemia cell lines. Although SLIT1 mRNA was scarce in un-stimulated BMMCs from healthy volunteers, SLIT1 gene expression increased 2.5–3.5 fold when incubated in conditioned medium from PHA or LPS stimulated peripheral blood mononuclear cells. The steady state expression level of SLIT1 by BMMCs from AA patients was 6 fold higher than from healthy individuals. SLIT1 mRNA was also detected in several myeloid leukemia cell lines including K562 and MV4-11, and its expression increased 2 fold by culturing the cells for 48 hours with the human stromal cell line HS-5. Examination of Robo receptor expression using antibodies specific to Robo1-4 showed that BM CD34+ cells from healthy individuals and K562 cells expressed Robo1. Recombinant Slit1 inhibited proliferation of K562 in a dose-dependent manner at concentrations of 1 and 5 mg/ml (Figure 1). Conclusions:The presence of clonal leukocytes with SLIT1 mutations in patients with immune-mediated AA suggests that HSCs with these mutations have survival or proliferation advantages over HSCs without SLIT1 mutations. Slit1 secreted from HSCs or hematopoietic progenitor cells in response to inflammatory cytokines may inhibit their own activation by binding to Robo1, but mutant Slit1 may fail to inhibit activation, leading to preferential commitment of the mutant HSCs to hematopoiesis. The Slit-Robo pathway may therefore serve as a novel therapeutic target for immune-mediated BM failure. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression of Roundabout Receptor 4 (Robo-4) Confers a Negative Prognosis On Patients with Acute Myeloid Leukemia

AbstractAbstract 1412 Background:Roundabout receptors – predominantly Robo-1 and Robo-4 – have been described as endothelial-specific markers which may be involved in the establishment of tumor neoangiogenesis. Since hematopoietic and endothelial cells are derived from a common precursor, the hemangioblast, they share many surface antigens such as CD34 or receptors for vascular endothelial growth factor. In this regard, expression of Robo-4 has been demonstrated on hematopoietic stem cells (HSC). It may serve here in cooperation with CXCR4 as an anchor for HSCs within the stem cell niche (Smith-Berdan et al, Cell Stem Cell. 2011;8(1):72–83).Similar to the normal hematopoietic system, leukemia is believed to derive from a leukemic stem cell (LSC) that serves as reservoir for leukemia cell renewal. Evidence exists that regulation of LSC biology is similar to that of normal hematopoietic stem cells (HSC).Therefore, the aim of our study was to investigate the expression of Roundabout family members Robo-1, Robo-4 and their ligand Slit-2 in primary AML samples followed by correlation to clinical data. Methods and Results:Pre-treatment samples from 104 patients with newly diagnosed AML who were entered into the AMLSG 07-04 trial were analyzed. The trial was conducted between August 2004 and August 2009 (ClinicalTrials.gov Identifier: NCT00151242). In brief, the trial design was as follows: Patients between 18 and 60 years of age with de novo or secondary AML [excluding patients with t(15;17)] were entered into the study. All patients received two induction courses with cytarabine, idarubicin and etoposide. Patients with complete remission (CR) obtained high dose cytarabine consolidation. At study entry patients were randomized between additional therapy with valproic acid, ATRA, both or none. After an interim analysis the randomization to the valproic acid and the combination arm was suspended.M-RNA expression values of Robo-1, Robo-4 and Slit-2 were determined in quantitative PCR analysis and normalized to expression of Glyceraldehyde 3-phosphate dehydrogenase. M-RNA of roundabout receptor Robo-4 was expressed in the majority of AML patients (83%) while expression of receptor Robo-1 was only detected in 27% of AML patients. Eighteen percent of AML patients showed expression of ligand Slit-2.In order to analyze the prognostic impact of gene expression levels, gene expression variables were categorized into two groups guided by conditional interference tree models for overall survival (Hothorn et al, J Comp Graph Stat, 2006; 15(3):651–674). Since no reasonable cut-off level for group separation was found for Robo-1 and Slit-2, the cohort was sub-divided in expressors and non-expressors for both gene expression variables.Clinical outcome was correlated to gene expression variables. For Robo-1 and Slit-2, no significant impact on survival could be observed. However, high Robo-4 expression (cut-off >0.095) had a significant impact on overall (OS) and event-free survival (EFS; p=0.002 and p=0.046, respectively; figure 1). The CR rate was 54% for the high Robo-4 expression group compared to 71% in the low Robo-4 expression group (p=0.103).Primary AML samples were analyzed for Robo-4 protein expression using flow cytometry (n=42). Robo-4 protein expression was observed in 81% of AML patients, although in most cases the expression was restricted to a very small proportion of AML cells. Double-staining for CD34 and Robo-4 revealed that a proportion of CD34+ showed co-expression of Robo-4 ranging from 0% to 22% of CD34+/Robo-4+ cells supporting that Robo-4 expression is restricted to a distinct CD34-sub-population possibly representing the LSC population. Conclusion:In conclusion, Robo-4 represents a negative prognostic marker in AML and therapeutic approaches against Robo-4 might preferentially target leukemic stem cells. [Display omitted] Disclosures:Fiedler:Pfizer Inc.: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

RabGDI controls axonal midline crossing by regulating Robo1 surface expression

BackgroundAxons navigate to their future synaptic targets with the help of choice points, intermediate targets that express axon guidance cues. Once they reach a choice point, axons need to switch their response from attraction to repulsion in order to move on with the next stage of their journey. The mechanisms underlying the change in axonal responsiveness are poorly understood. Commissural axons become sensitive to the repulsive activity of Slits when they cross the ventral midline of the CNS. Responsiveness to Slits depends on surface expression of Robo receptors. In Drosophila, Commissureless (Comm) plays a crucial regulatory role in midline crossing by keeping Robo levels low on precommissural axons. Interestingly, to date no vertebrate homolog of comm has been identified. Robo3/Rig1 has been shown to control Slit sensitivity before the midline, but without affecting Robo1 surface expression.ResultsWe had identified RabGDI, a gene linked to human mental retardation and an essential component of the vesicle fusion machinery, in a screen for differentially expressed floor-plate genes. Downregulation of RabGDI by in ovo RNAi caused commissural axons to stall in the floor plate, phenocopying the effect observed after downregulation of Robo1. Conversely, premature expression of RabGDI prevented commissural axons from entering the floor plate. Furthermore, RabGDI triggered Robo1 surface expression in cultured commissural neurons. Taken together, our results identify RabGDI as a component of the switching mechanism that is required for commissural axons to change their response from attraction to repulsion at the intermediate target.ConclusionRabGDI takes over the functional role of fly Comm by regulating the surface expression of Robo1 on commissural axons in vertebrates. This in turn allows commissural axons to switch from attraction to repulsion at the midline of the spinal cord.

Gbx2 regulates thalamocortical axon guidance by modifying the LIM and Robo codes.

Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs.

Activation of Robo1 Signaling of Breast Cancer Cells by Slit2 from Stromal Fibroblast Restrains Tumorigenesis via Blocking PI3K/Akt/β-Catenin Pathway

Tumor microenvironment plays a critical role in regulating tumor progression by secreting factors that mediate cancer cell growth. Stromal fibroblasts can promote tumor growth through paracrine factors; however, restraint of malignant carcinoma progression by the microenvironment also has been observed. The mechanisms that underlie this paradox remain unknown. Here, we report that the tumorigenic potential of breast cancer cells is determined by an interaction between the Robo1 receptor and its ligand Slit2, which is secreted by stromal fibroblasts. The presence of an active Slit2/Robo1 signal blocks the translocation of β-catenin into nucleus, leading to downregulation of c-myc and cyclin D1 via the phosphoinositide 3-kinase (PI3K)/Akt pathway. Clinically, high Robo1 expression in the breast cancer cells correlates with increased survival in patients with breast cancer, and low Slit2 expression in the stromal fibroblasts is associated with lymph node metastasis. Together, our findings explain how a specific tumor microenvironment can restrain a given type of cancer cell from progression and show that both stromal fibroblasts and tumor cell heterogeneity affect breast cancer outcomes.

Slits and Robo-2 regulate the coalescence of subsets of olfactory sensory neuron axons within the ventral region of the olfactory bulb

Olfactory sensory neurons (OSNs) project their axons to second-order neurons in the olfactory bulb (OB) to form a precise glomerular map and these stereotypic connections are crucial for accurate odorant information processing by animals. To form these connections, olfactory sensory neuron (OSN) axons respond to axon guidance molecules that direct their growth and coalescence. We have previously implicated the axon guidance receptor Robo-2 in the accurate coalescence of OSN axons within the dorsal region of the OB (Cho et al., 2011). Herein, we have examined whether Robo-2 and its ligands, the Slits, contribute to the formation of an accurate glomerular map within more ventral regions of the OB. We have ablated expression of Robo-2 in OSNs and assessed the targeting accuracy of axons expressing either the P2 or MOR28 olfactory receptors, which innervate two different regions of the ventral OB. We show that P2-positive axons, which express Robo-2, coalesce into glomeruli more ventrally and form additional glomeruli in the OB of robo-2lox/lox;OMP-Cre mice. We also demonstrate that Robo-2-mediated targeting of P2 axons along the dorsoventral axis of the OB is controlled by Slit-1 and Slit-3 expression. Interestingly, although MOR28-positive OSNs only express low levels of Robo-2, a reduced number of MOR28-positive glomeruli is observed in the OB of robo-2lox/lox;OMP-Cre mice. Taken together, our results demonstrate that Slits and Robo-2 are required for the formation of an accurate glomerular map in the ventral region of the OB.

Slit2 and Robo3 modulate the migration of GnRH-secreting neurons

Gonadotropin-releasing hormone (GnRH) neurons are born in the nasal placode and migrate along olfactory and vomeronasal axons to reach the forebrain and settle in the hypothalamus, where they control reproduction. The molecular cues that guide their migration have not been fully identified, but are thought to control either cell movement directly or the patterning of their axonal substrates. Using genetically altered mouse models we show that the migration of GnRH neurons is directly modulated by Slit2 and Robo3, members of the axon guidance Slit ligand and Robo receptor families. Mice lacking Slit2 or Robo3 have a reduced number of GnRH neurons in the forebrain, but a normal complement of their supporting axons, pointing to a direct role for these molecules in GnRH neuron migration.

Fragments of SLIT3 inhibit cellular migration

The repellent factor family of Slit molecules has been described as having a repulsive function in the developing nervous system on growing axons expressing the Roundabout (Robo) receptors. Recent studies determined the effects of Slit molecules on the migratory and invasive potential of several types of tumor cells but also on synovial fibroblasts (SFs) derived from rheumatoid arthritis (RA) patients. To optimize a potential therapeutic application we aimed at generatingfragments of Slit3 showing the same functional ability as the full-length molecule but having the advantage of a smaller size. Recombinant Slit3 proteins were expressed and analyzed by western blotting. Their activity was defined by functional assays such as migration assays with RASF and melanoma cells. Recombinant Slit3 containing only leucine rich repeat domain 2 (D2), the domain important for Robo binding and the minimal functional unit D2 dNC were both able to inhibit migration of RASFs as effectively as Slit3 with all 4 repeats. Collectively, our data showed that the ability of Slit3 to reduce the migratory activity of synovial cells from patients with RA and melanoma cells can be mimicked by small protein fragments derived from Slit3. Slit3 fragments may be helpful in therapeutic attempts; however, further studies are necessary in order to elucidate their activity in vivo.

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1–3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34+ HSPCs. Human CD34+ HSPCs expressed mRNAs for all Robos (Robo1–4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34+ HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34+HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34+HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34+ HSPCs.

The Actin-Binding Protein Canoe/AF-6 Forms a Complex with Robo and Is Required for Slit-Robo Signaling during Axon Pathfinding at the CNS Midline

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.

Slits affect the timely migration of neural crest cells via Robo receptor.

Neural crest cells emerge by delamination from the dorsal neural tube and give rise to various components of the peripheral nervous system in vertebrate embryos. These cells change from non-motile into highly motile cells migrating to distant areas before further differentiation. Mechanisms controlling delamination and subsequent migration of neural crest cells are not fully understood. Slit2, a chemorepellant for axonal guidance that repels and stimulates motility of trunk neural crest cells away from the gut has recently been suggested to be a tumor suppressor molecule. The goal of this study was to further investigate the role of Slit2 in trunk neural crest cell migration by constitutive expression in neural crest cells. We found that Slit gain-of-function significantly impaired neural crest cell migration while Slit loss-of-function favored migration. In addition, we observed that the distribution of key cytoskeletal markers was disrupted in both gain and loss of function instances. These findings suggest that Slit molecules might be involved in the processes that allow neural crest cells to begin migrating and transitioning to a mesenchymal type.

Morphological phenotypes of olfactory ensheathing cells display different migratory responses upon Slit-2

Olfactory ensheathing cells (OECs) are a type of glial cells with morphological plasticity in the olfactory system. Cultured OECs display the process-bearing and flattened shape. Our previous studies have shown that the frontal application of Slit-2 gradient induced the collapse of leading front, and reversed the soma translocation of process-bearing OECs. However, the migratory properties of flattened OECs upon Slit-2 gradient remain elusive. Here, we found that Slit-2 gradient induced the collapse of their plasma membrane, and inhibited migration of flattened OECs. Upon to Slit-2 gradient, the leading front of flattened type 1 OECs firstly showed collapse and retraction, then gradually re-grew a new lamellipodia, finally, showed collapse again (this phenomenon was called as adaptation), while flattened type 2 OECs only showed collapse of plasma membrane. These different migratory responses upon Slit-2 stimulation were possibly due to their different sub-cellular distribution of Robo receptor. Furthermore, F-actin at the peripheral region of leading front was more sensitive to the Slit-2 stimulation than microtubules and the loss of F-actin might be implicated in initiating the collapse of flattened OECs. Finally, the adaptation of flattened type 1 OECs induced by Slit-2 was independent on protein synthesis. Taken together, these results demonstrate that morphological phenotypes of OECs display different migratory properties upon Slit-2 and an unexpected finding that the protein synthesis-independent adaptation in OECs induced by Slit-2.

Robo1 Regulates the Migration and Laminar Distribution of Upper-Layer Pyramidal Neurons of the Cerebral Cortex

Laminar organization is a key feature of the mammalian cerebral cortex, but the mechanisms by which final positioning and "inside-out" distribution of neurons are determined remain largely unknown. Here, we demonstrate that Robo1, a member of the family of Roundabout receptors, regulates the correct positioning of layers II/III pyramidal neurons in the neocortex. Specifically, we used RNA interference in mice to suppress the expression of Robo1 in a subset of layers II/III neurons, and observed the positions of these cells at distinct developmental stages. In contrast to control neurons that migrated toward the pial surface by P1, Robo1-suppressed neurons exhibited a delay in entering the cortical plate at respective stages. Unexpectedly, after the first postnatal week, these neurons were predominantly located in the upper part of layers II/III, in contrast to control cells that were distributed throughout these layers. Sequential electroporation studies revealed that Robo1-suppressed cells failed to establish the characteristic inside-out neuronal distribution and, instead, they accumulated beneath the marginal zone regardless of their birthdate. These results demonstrate that Robo receptors play a crucial role in neocortical lamination and particularly in the positioning of layers II/III pyramidal neurons.

Slit-Robo: A potential way to treat periodontitis

Slit is a secreted protein known to function through the Roundabout (Robo) receptor. Slit has recently been shown to be an endogenously available inhibitor of leukocyte chemotaxis and as a chemoattractant to recruit vascular endothelial cells to sites for angiogenesis both in vivo and in vitro. The initiation and progression of periodontal diseases, is the result of complex interactions between the colonizing bacteria in the periodontal pockets and host immune and inflammatory responses. Antibiotics such as tetracyclines are commonly used in the management of periodontal infections and yet, have shown modest success in reducing neutrophil-mediated injury. Angiogenesis is important for the maintenance of homeostatus of periodontal tissues. However, few studies have been reported about angiogenesis targeted treatment for periodontitis. Based on its angiogenesis promoting effect and leukocyte chemotaxis inhibition effect, we hypothesize that Slit can be an effective immunotherapeutic agent in the treatment of periodontitis.

The role of Slit‐Robo signaling in Congenital Diaphragmatic Hernia

Congenital Diaphragmatic Hernia (CDH) is a common birth defect associated with high mortality and severe respiratory deficiency in survivors. A general feature of CDH is the herniation of the abdominal tissues into the chest cavity, leading to malformation of the lung. The cellular and molecular mechanisms underlying CDH remain poorly understood. We found that mice deficient in the function of two Roundabout (Robo) receptors, Robo1 and Robo2, exhibit defects resembling CDH. Determination of the origin of these defects led to an unexpected role of Robo in the separation of foregut from the dorsal body wall, a morphogenetic process that is vital, yet rarely studied. Together, these data suggest for the first time that delayed separation of the foregut tissues from the dorsal body wall may contribute to the etiology of CDH in humans.

TheLhx2Transcription Factor Controls Thalamocortical Axonal Guidance by Specific Regulation of Robo1 and Robo2 Receptors

The assembly of neural circuits is dependent upon the generation of specific neuronal subtypes, each subtype displaying unique properties that direct the formation of selective connections with appropriate target cells. Actions of transcription factors in neural progenitors and postmitotic cells are key regulators in this process. LIM-homeodomain transcription factors control crucial aspects of neuronal differentiation, including subtype identity and axon guidance. Nonetheless, their regulation during development is poorly understood and the identity of the downstream molecular effectors of their activity remains largely unknown. Here, we demonstrate that the Lhx2 transcription factor is dynamically regulated in distinct pools of thalamic neurons during the development of thalamocortical connectivity in mice. Indeed, overexpression of Lhx2 provokes defective thalamocortical axon guidance in vivo, while specific conditional deletion of Lhx2 in the thalamus produces topographic defects that alter projections from the medial geniculate nucleus and from the caudal ventrobasal nucleus in particular. Moreover, we demonstrate that Lhx2 influences axon guidance and the topographical sorting of axons by regulating the expression of Robo1 and Robo2 guidance receptors, which are essential for these axons to establish correct connections in the cerebral cortex. Finally, augmenting Robo1 function restores normal axon guidance in Lhx2-overexpressing neurons. By regulating axon guidance receptors, such as Robo1 and Robo2, Lhx2 differentially regulates the axon guidance program of distinct populations of thalamic neurons, thus enabling the establishment of specific neural connections.

Multiple Slits regulate the development of midline glial populations and the corpus callosum

The Slit molecules are chemorepulsive ligands that regulate axon guidance at the midline of both vertebrates and invertebrates. In mammals, there are three Slit genes, but only Slit2 has been studied in any detail with regard to mammalian brain commissure formation. Here, we sought to understand the relative contributions that Slit proteins make to the formation of the largest brain commissure, the corpus callosum. Slit ligands bind Robo receptors, and previous studies have shown that Robo1−/− mice have defects in corpus callosum development. However, whether the Slit genes signal exclusively through Robo1 during callosal formation is unclear. To investigate this, we compared the development of the corpus callosum in both Slit2−/− and Robo1−/− mice using diffusion magnetic resonance imaging. This analysis demonstrated similarities in the phenotypes of these mice, but crucially also highlighted subtle differences, particularly with regard to the guidance of post-crossing axons. Analysis of single mutations in Slit family members revealed corpus callosum defects (but not complete agenesis) in 100% of Slit2−/− mice and 30% of Slit3−/− mice, whereas 100% of Slit1−/−; Slit2−/− mice displayed complete agenesis of the corpus callosum. These results revealed a role for Slit1 in corpus callosum development, and demonstrated that Slit2 was necessary but not sufficient for midline crossing in vivo. However, co-culture experiments utilising Robo1−/− tissue versus Slit2 expressing cell blocks demonstrated that Slit2 was sufficient for the guidance activity mediated by Robo1 in pre-crossing neocortical axons. This suggested that Slit1 and Slit3 might also be involved in regulating other mechanisms that allow the corpus callosum to form, such as the establishment of midline glial populations. Investigation of this revealed defects in the development and dorso-ventral positioning of the indusium griseum glia in multiple Slit mutants. These findings indicate that Slits regulate callosal development via both classical chemorepulsive mechanisms, and via a novel role in mediating the correct positioning of midline glial populations. Finally, our data also indicate that some of the roles of Slit proteins at the midline may be independent of Robo signalling, suggestive of additional receptors regulating Slit signalling during development.

Autocrine/juxtaparacrine regulation of axon fasciculation by Slit-Robo signaling

Axons travel to their targets in bundles or fascicles, but the molecules regulating fasciculation remain incompletely characterized. We found that Slit2 and its Robo receptors are expressed by motor axons, and that inactivation of Slit2 or Robo1 and Robo2 in mice caused axons to defasciculate prematurely at muscle targets. In vitro, Slit2 secreted by motoneurons regulated fasciculation through Robo1 and Robo2. These results support the idea that Slit2 promotes axon fasciculation via an autocrine and/or juxtaparacrine mechanism.

Maintaining Muscle Mitochondria via Transsynaptic Signaling

Dominant VAPB mutations are implicated in neurodegenerative disease, including amyotrophic lateral sclerosis and spinal muscular atrophy. In the current issue, Han et al. (2012) uncover a mechanism through which the secreted VAPB MSP domain regulates actin organization and mitochondrial function in muscle cells through LAR and Robo receptor activation.