Repulsive Signals Research Articles

Light-Activated Gene Expression Directs Segregation of Co-cultured Cells in Vitro

Light-directed gene patterning methods have been described as a means to regulate gene expression in a spatially and temporally controlled manner. Several methods have been reported that use photocaged forms of small molecule effectors to control ligand-dependent transcription factors. Whereas these methods offer many advantages including high specificity and transient light-sensitivity, the free diffusion of the uncaged effector can limit both the magnitude and resolution of localized gene induction. Methods to date have been limited by the small fraction of irradiated cells that have expression levels significantly above uninduced background and have not been shown to affect a defined biological response. The tetracycline-dependent transactivator/transrepressor system, RetroTET-ART, combined with a photocaged form of doxycycline (NvOC-Dox) can be used to form photolithographic patterns of induced expression wherein up to 85% of the patterned cells show expression levels above uninduced regions. The efficiency and inducibility of the RetroTET-ART system allows one to quantitatively measure the limits of resolution and the relative induction levels mediated by a small molecule photocaged effector for the first time. Well-defined patterns of reporter genes were reproducibly formed within 6-36 h with feature sizes as small as 300 microm. After photo-patterning, NvOC-Dox can be rapidly removed, rendering cells photoinsensitive and allowing one to monitor GFP product formation in real time. Patterned co-expression of the cell surface ligand ephrin A5 on cell monolayers creates well-defined patterns that are sufficient to direct and segregate co-cultured cells via either attractive or repulsive signaling cues. The ability to direct the arrangement of cells on living cell monolayers through the action of light may serve as a model system for engineering artificial tissues.

Dissociation of EphB2 Signaling Pathways Mediating Progenitor Cell Proliferation and Tumor Suppression

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

Guiding Axons in the Central Nervous System: A Tissue Engineering Approach

Complete axon regeneration after trauma or disease is largely unsuccessful in the central nervous system. With the fast developing advances in tissue engineering and biomaterials, many investigations have identified promising approaches for guiding axonal extension. This review highlights a variety of these approaches and describes the biomaterial properties and signaling mechanisms involved in the fabrication of optimal guidance platforms. The vast majority of axonal regeneration approaches limit themselves to observe how axons elongate and migrate in response to signaling molecules presented on the substrate materials, or more recently, in response to different chemical and mechanical substrate properties. Many of these studies are encouraging in the hope of regenerating axons after disease or injury; however, numerous barriers remain. Here we illustrate the need to optimize a permissive heterogeneous environment for axon elongation using tissue engineering approaches and a thorough understanding of the mechanical properties of the substrate, mechanotaxis, and both attractive and repulsive signaling mechanisms.

Antagonistic interactions among Plexins regulate the timing of intersegmental vessel formation

The angioblast is an embryonic endothelial cell precursor that migrates long distances to reach its final position, navigating by sensing attractive and repulsive cues from the environment. Members of the semaphorin family have been implicated in controlling the behaviour of angioblast tip cells through repulsive signalling in vitro, but their in vivo roles are less clear. Here we show that zebrafish semaphorin3e (sema3e) is expressed by endothelial cells of the dorsal aorta, primary motoneurons, and endodermal cells. Further, loss of Sema3e leads to delayed exit of angioblasts from the dorsal aorta in ISV formation. Through transplant analysis, we show that Sema3e acts autonomously and non-autonomously in angioblasts to modulate interactions among themselves. The semaphorin receptors, PlexinD1 and PlexinB2, are expressed by zebrafish angioblasts. Loss of plxnB2 results in delayed ISV sprouting identical to that seen in sema3e morphants, while loss of plexinD1 in out of bounds (obd) mutants results in precocious ISV sprouting. Loss of either sema3e or plxnB2 in obd mutants generates an intermediate phenotype, suggesting that PlxnD1 and Sema3e/PlxnB2 antagonize each other to control timing of ISV sprouting. Consistent with this observation, we show that PlxnB2 acts cell autonomously in endothelial cells. This suggests a model where multiple semaphorin–plexin interactions control angioblast sprouting behaviour.

Robo1/Robo4: Different expression patterns in retinal development

Two members of the roundabout (Robo) family, Robo1 and Robo4, serve as neuronal guidance receptors. During neurogenesis, Robo1 and Robo4 participate in axonal guidance by mediating a repulsive signal. It has been reported that Robo4 is mainly expressed in the vasculature and that Robo1 is expressed both in neural and non-neural tissues. However, the roles of these Robo proteins in the mammalian vasculature are still unclear. In this current study, the expression patterns of Robo1 and Robo4 in the retinal vasculature were determined using C57BL/6J mice at postnatal days (P) 1, P3, P5, P7, P9, P12, P14, P17, P21 and adult mice (1month). We found that Robo4 was expressed not only in the retinal vessels but also in the retinal ganglion cell and photoreceptor layers during retinal development. Robo4 expression peaked at P3 and P9, which suggest that Robo4 may function in stabilizing the retinal vasculature. Robo1 expression was observed in the retina neuronal cells and vessels. Both Robo1 mRNA and protein expression showed a typical expression pattern, which related to Robo1’s roles in the different stages of retinal vascular development in the murine retina. Robo1 displayed high expression levels at P1 (correlated with superficial vascular plexus formation) and P7 (correlated with deep vascular plexus formation). The high levels of Robo1 during these two well-defined phases of retinal capillary plexus formation indicate that Robo1 is likely to play a part in retinal neovascularization.

Contactin is a signal receptor for the notochord-derived repulsive guidance signal in Xenopus primary sensory axons

International Journal of Developmental NeuroscienceVolume 26, Issue 8 p. 882-883 Article [P2.48]: Contactin is a signal receptor for the notochord-derived repulsive guidance signal in Xenopus primary sensory axons S. Nagata, Corresponding Author S. Nagata n/a@.dne Japan Women's University, JapanCorresponding author.Search for more papers by this authorN. Fujita, N. Fujita Japan Women's University, JapanSearch for more papers by this author S. Nagata, Corresponding Author S. Nagata n/a@.dne Japan Women's University, JapanCorresponding author.Search for more papers by this authorN. Fujita, N. Fujita Japan Women's University, JapanSearch for more papers by this author First published: 25 November 2008 https://doi.org/10.1016/j.ijdevneu.2008.09.173Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume26, Issue8Abstracts to the 17th Biennial Meeting of the International Society for Developmental Neuroscience, 1-5 June 2008, Asilomar, USADecember 2008Pages 882-883 RelatedInformation

Ephrin-B2-induced Cleavage of EphB2 Receptor Is Mediated by Matrix Metalloproteinases to Trigger Cell Repulsion

EphB receptors provide crucial adhesive and repulsive signals during cell migration and axon guidance, but it is unclear how they switch between these opposing responses. Here we provide evidence of an important role for matrix metalloproteinases (MMPs) in repulsive EphB2 signaling. We found that EphB2 is cleaved by MMPs both in vitro and in vivo, and that this cleavage is induced by interaction with its ligand ephrin-B2. Our findings demonstrate that MMP-2/MMP-9-specific inhibition or cleavage-resistant mutations in the ectodomain of EphB2 can prevent EphB2-mediated cell-cell repulsion in HEK293 cells, and block ephrin-B1-induced growth cone withdrawal in cultured hippocampal neurons. Transient expression of wtEphB2, but not noncleavable EphB2-4/5 mutant, restored ephrin-B1-induced growth cone collapse and withdrawal in EphB-deficient neurons. The inhibition of EphB2 cleavage also had potent regulatory effects on EphB2 activity. This study provides the first evidence that MMP-mediated cleavage of EphB2 is induced by receptor-ligand interactions at the cell surface and that this event triggers cell-repulsive responses.

BAG1 promotes axonal outgrowth and regeneration in vivo via Raf-1 and reduction of ROCK activity

Improved survival of injured neurons and the inhibition of repulsive environmental signalling are prerequisites for functional regeneration. BAG1 (Bcl-2-associated athanogene-1) is an Hsp70/Hsc70-binding protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. We investigated BAG1 as a therapeutic molecule in the lesioned visual system in vivo. Using an adeno-associated viral vector, BAG1 (AAV.BAG1) was expressed in retinal ganglion cells (RGC) and then tested in models of optic nerve axotomy and optic nerve crush. BAG1 significantly increased RGC survival as compared to adeno-associated viral vector enhanced green fluorescent protein (AAV.EGFP) treated controls and this was independently confirmed in transgenic mice over-expressing BAG1 in neurons. The numbers and lengths of regenerating axons after optic nerve crush were also significantly increased in the AAV.BAG1 group. In pRGC cultures, BAG1-over-expression resulted in a approximately 3-fold increase in neurite length and growth cone surface. Interestingly, BAG1 induced an intracellular translocation of Raf-1 and ROCK2 and ROCK activity was decreased in a Raf-1-dependent manner by BAG1-over-expression. In summary, we show that BAG1 acts in a dual role by inhibition of lesion-induced apoptosis and interaction with the inhibitory ROCK signalling cascade. BAG1 is therefore a promising molecule to be further examined as a putative therapeutic tool in neurorestorative strategies.

Vascular Endothelial Growth Factor and Semaphorin Induce Neuropilin-1 Endocytosis via Separate Pathways

The neuropilin (Nrp)1 receptor is essential for both nervous and vascular system development. Nrp1 is unusually versatile, because it transmits both chemoattractive and repulsive signals in response to vascular endothelial growth factor (VEGF)-A and class 3 semaphorins, respectively. Both Nrp1 and VEGF receptor 2 undergo ligand-dependent endocytosis. We sought to establish the endocytic pathway of Nrp1 and to determine whether uptake is required for its signaling. Whereas Nrp1 underwent clathrin-dependent endocytosis in response to VEGFA(165) treatment, semaphorin 3C (sema3C) induced lipid raft-dependent endocytosis. The myosin VI PDZ (postsynaptic density 95, Disk large, Zona occludens-1) adaptor protein synectin was essential for Nrp1 trafficking. Sema3C failed to inhibit migration of synectin(-/-) endothelial cells, mirroring the lower migratory response of these cells to VEGFA(165). These results show that the endocytic pathway of Nrp1 is determined by its ligand and that the trafficking of Nrp1 is essential for its signaling.

Repulsive Wnt Signaling Inhibits Axon Regeneration after CNS Injury

Failure of axon regeneration in the mammalian CNS is attributable in part to the presence of various inhibitory molecules, including myelin-associated proteins and proteoglycans enriched in glial scars. Here, we evaluate whether axon guidance molecules also regulate regenerative growth after injury in adulthood. Wnts are a large family of axon guidance molecules that can attract ascending axons and repel descending axons along the length of the developing spinal cord. Their expression (all 19 Wnts) is not detectable in normal adult spinal cord by in situ hybridization. However, three of them are clearly reinduced after spinal cord injury. Wnt1 and Wnt5a, encoding potent repellents of the descending corticospinal tract (CST) axons, were robustly and acutely induced broadly in the spinal cord gray matter after unilateral hemisection. Ryk, the conserved repulsive Wnt receptor, was also induced in the lesion area, and Ryk immunoreactivity was found on the lesioned CST axons. Wnt4, which attracts ascending sensory axons in development, was acutely induced in areas closer to the lesion than Wnt1 and Wnt5a. Injection of function-blocking Ryk antibodies into the dorsal bilateral hemisectioned spinal cord either prevented the retraction of CST axons or promoted their regrowth but clearly enhanced the sprouting of CST collateral branches around and beyond the injury site. Therefore, repulsive Wnt signaling may be a cause of cortical spinal tract axon retraction and inhibits axon sprouting after injury.

Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin–mediated cell adhesion during Drosophila heart tube lumen formation

During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad–mediated cell adhesion.

Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.

Cyclic GMP-Gated CNG Channels Function in Sema3A-Induced Growth Cone Repulsion

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. We show that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. We also show that Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend our knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance.

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision.

Cross‐midline interactions between mouse commissural hindbrain axons contribute to their efficient decussation

Information from both sides of the brain is integrated by axons that project across the midline of the central nervous system via numerous commissures present at all axial levels. Despite the accumulated experimental evidence, questions remain regarding the formation of commissures in the presence of strong repulsive signals in the ventral midline. Studies from invertebrates suggest that interaction at the midline between homologous axons of specific decussating neurons contributes to efficient midline crossing, but such evidence is lacking in vertebrate systems. We performed experiments to determine whether commissural axons of the caudal region of the hindbrain interact with their contralateral counterparts at the ventral midline and to evaluate the relevance of this reciprocal interaction. Double anterograde axon labeling with lipophilic tracers revealed close apposition between growth cones of contralateral pioneer decussating axons at the midline. Later, we detected fasciculation between contralateral axons that is maintained even after they have crossed the midline. Blocking axon projections unilaterally with a solid mechanical barrier decreased dramatically the midline crossing of the equivalent population from the contralateral side. Decussation was also blocked by a unilateral barrier permeable to diffusible molecules but not by an axon-permeable barrier. These results suggest that in the caudal region of the hindbrain, midline crossing is facilitated by interactions between decussating contralateral axon partners.

Gating of Sema3E/PlexinD1 Signaling by Neuropilin-1 Switches Axonal Repulsion to Attraction during Brain Development

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. Here, we show 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. Our data therefore reveal a "gating" function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.

The Mechanisms Underlying Primitive Streak Formation in the Chick Embryo

Formation of the primitive streak is one of the key events in the early development of amniote embryos. The streak is the site where during gastrulation the mesendoderm cells ingress to take up their correct topographical positions in the embryo. The process of streak formation can be conveniently observed in the chick embryo, where the streak forms as an accumulation of cells in the epiblast in the posterior pole of the embryo and extends subsequently in anterior direction until it covers 80% of the epiblast. A prerequisite for streak formation is the differentiation of mesoderm, which is induced in the epiblast at the interface between the posterior Area Opaca and Area Pellucida in a sickle shaped domain overlying Koller's sickle. Current views on the molecular mechanisms of mesoderm induction by inducing signals from the Area Opaca and inhibitory signals from the hypoblast are briefly discussed. During streak formation the sickle of mesoderm cells transforms into an elongated structure in the central midline of the embryo. We discuss possible cellular mechanisms underlying this process, such as oriented cell division, cell-cell intercalation, chemotactic cell movement in response to attractive and repulsive signals and a combination of chemotaxis and contact following. We review current experimental evidence in favor and against these different hypotheses and outline some the outstanding questions. Since many of the interactions between cells signaling and moving are dynamic and nonlinear in nature they will require detailed modeling and computer simulations to be understood in detail.

Neuronal Pathfinding

During development, the growing tips of neurons must locate and identify their preferred targets. Neurons follow attractive and repulsive guidance signals to build a network to connect muscle activity to brain signals, sensory inputs to behavioral responses, and so on. Fujisawa et al . have now discovered another component, EVA-1, in the guidance system for neurons that fine-tunes the guidance signals sent and received by different neurons in the model organism Caenorhabditis elegans . K. Fujisawa, J. L. Wrana, J. G. Culotti, The Slit receptor EVA-1 coactivates a SAX-3/Robo–mediated guidance signal in C. elegans . Science 317 , 1934-1938 (2007). [Abstract] [Full Text]

Different Wnt signals act through the Frizzled and RYK receptors duringDrosophilasalivary gland migration

Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Here we show that two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. Our results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration.

A Motor for Netrin Receptors

Netrin receptors include neogenin and DCC (deleted in colorectal cancer). These receptors function in axon guidance, with neogenin implicated in the response to repulsive guidance cues and DCC in the response to attractive guidance cues. Myosin X (Myo X) is an unconventional myosin implicated in the formation of cellular projections. Zhu et al . found that Myo X interacted with neogenin and DCC in a manner that required the FERM domain of Myo X and the P3 region of the netrin receptor. Neogenin interacted more strongly with Myo X lacking the motor domain, whereas DCC showed a preference for full-length Myo X. (A motorless form of Myo X generated by alternative splicing has been reported in brain.) Overexpression studies in COS7 cells, rat cortical neurons, and the neuronal cell line NLT suggested that Myo X and DCC colocalized at the tips of filopodia and that Myo X increased the delivery of DCC to these structures. RNA interference studies to knock down Myo X in NLT cells showed that, in the absence of endogenous Myo X, DCC targeting to neurites was decreased (based on immunofluorescence analysis). NLT cells exhibited opposing responses to netrin depending on whether MyoX was coexpresssed with neogenin or DCC. Netrin treatment of the DCC- and Myo X-expressing cells resulted in the formation of neurites and a neuronal-like morphology, whereas the neogenin- and Myo X-expressing cells adopted a spread morphology lacking filopodia or neurites. The motorless Myo X was overexpressed in mouse embryos using in utero electroporation, and the response of cortical explants from these mice to netrin was analyzed. The neurons expressing the motorless Myo X showed shorter neurites in the absence of netrin and reduced outgrowth in response to netrin. In ovo electroporation of the motorless Myo X allowed the effect of Myo X on commissural axon guidance to be analyzed in chicken embryos. Neurons expressing the motorless Myo X failed to cross the midline. This study demonstrates a direct association between a motor protein, Myo X, and the plasma membrane netrin receptors. Furthermore, the differential interaction of neogenin with a motorless form of Myo X, which appears to inhibit neurite extension and axon guidance, provides a potential mechanism for repulsive signaling by neogenin. X.-J. Zhu, C.-Z. Wang, P.-G. Dai, Y. Xie, N.-N. Song, Y. Liu, Q.-S. Du, L. Mei, Y.-Q. Ding, W.-C. Xiong, Myosin X regulates netrin receptors and functions in axonal path-finding. Nat. Cell Biol. 9 , 184-192 (2007). [PubMed]