Where does axon guidance lead us?

During neural circuit formation, axons need to navigate to their target cells in a complex, constantly changing environment. Although we most likely have identified most axon guidance cues and their receptors, we still cannot explain the molecular background of pathfinding for any subpopulation of axons. We lack mechanistic insight into the regulation of interactions between guidance receptors and their ligands. Recent developments in the field of axon guidance suggest that the regulation of surface expression of guidance receptors comprises transcriptional, translational, and post-translational mechanisms, such as trafficking of vesicles with specific cargos, protein-protein interactions, and specific proteolysis of guidance receptors. Not only axon guidance molecules but also the regulatory mechanisms that control their spatial and temporal expression are involved in synaptogenesis and synaptic plasticity. Therefore, it is not surprising that genes associated with axon guidance are frequently found in genetic and genomic studies of neurodevelopmental disorders

Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum

<p>Abstract</p> <p>Background</p> <p>Neural development consists of a series of steps, including neurogenesis, patterning, cell migration, axon guidance, and finally, synaptogenesis. Because all these steps proceed in a constantly changing environment, functional gene analyses during development have to take time into account. This is quite challenging, however, as loss-of-function approaches based on classic genetic tools do not allow for the precise temporal control that is required for developmental studies. Gene silencing by RNA interference (RNAi) in combination with the chicken embryo or with cultured embryos opens new possibilities for functional gene analysis <it>in vivo</it>. Axonin-1/TAG-1 is a cell adhesion molecule of the immunoglobulin superfamily with a well defined temporal and spatial expression pattern in the developing vertebrate nervous system. Axonin-1/TAG-1 was shown to promote neurite outgrowth <it>in vitro </it>and to be required for commissural and sensory axon pathfinding <it>in vivo</it>.</p> <p>Results</p> <p>To knock down axonin-1 in a temporally and spatially controlled manner during development of the nervous system, we have combined RNAi with the accessibility of the chicken embryo even at late stages of development. Using <it>ex ovo </it>RNAi, we analyzed the function of axonin-1/TAG-1 in cerebellar development. Axonin-1 is expressed in postmitotic granule cells while they extend their processes, the parallel fibers. In the absence of axonin-1 these processes still extend but no longer in a parallel manner to each other or to the pial surface of the cerebellum.</p> <p>Conclusion</p> <p>Axonin-1/TAG-1 is required for the navigation, but not for the elongation, of granule cell processes in the developing cerebellum <it>in vivo</it>.</p

Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development

Many genes have several, sometimes divergent functions during development. Therefore, timing of gene knockdown for functional analysis during development has to be done with precise temporal control, as loss of a gene's function at early stages prevents its analysis later in development. RNAi, in combination with the accessibility of chicken embryos, is an effective approach for temporally controlled analysis of gene function during neural development. Here, we describe novel plasmid vectors that contain cell type-specific promoters/enhancers to drive the expression of a fluorescent marker, followed directly by a miR30-RNAi transcript for gene silencing. These vectors allow for direct tracing of cells experiencing gene silencing by the bright fluorescence. The level of knockdown is sufficient to reproduce the expected pathfinding defects upon perturbation of genes with known axon guidance functions. Mixing different vectors prior to electroporation enables the simultaneous knockdown of multiple genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a fast and precise manner. The advancements of the in ovo RNAi technique that we describe will not only markedly enhance functional gene analysis in the chicken, but also could be adapted to other organisms in developmental studie

Understanding axon guidance: are we nearly there yet?

During nervous system development, neurons extend axons to reach their targets and form functional circuits. The faulty assembly or disintegration of such circuits results in disorders of the nervous system. Thus, understanding the molecular mechanisms that guide axons and lead to neural circuit formation is of interest not only to developmental neuroscientists but also for a better comprehension of neural disorders. Recent studies have demonstrated how crosstalk between different families of guidance receptors can regulate axonal navigation at choice points, and how changes in growth cone behaviour at intermediate targets require changes in the surface expression of receptors. These changes can be achieved by a variety of mechanisms, including transcription, translation, protein-protein interactions, and the specific trafficking of proteins and mRNAs. Here, I review these axon guidance mechanisms, highlighting the most recent advances in the field that challenge the textbook model of axon guidance

Rostral growth of commissural axons requires the cell adhesion molecule MDGA2

Background: Long-distance axonal growth relies on the precise interplay of guidance cues and cell adhesion molecules. While guidance cues provide positional and directional information for the advancing growth cone, cell adhesion molecules are essential in enabling axonal advancement. Such a dependence on adhesion as well as guidance molecules can be well observed in dorsal commissural interneurons, which follow a highly stereotypical growth and guidance pattern. The mechanisms and molecules involved in the attraction and outgrowth towards the ventral midline, the axon crossing towards the contralateral side, the rostral turning after midline crossing as well as the guidance along the longitudinal axis have been intensely studied. However, little is known about molecules that provide the basis for commissural axon growth along the anterior-posterior axis. Results: MDGA2, a recently discovered cell adhesion molecule of the IgCAM superfamily, is highly expressed in dorsolaterally located (dI1) spinal interneurons. Functional studies inactivating MDGA2 by RNA interference (RNAi) or function-blocking antibodies demonstrate that either treatment results in a lack of commissural axon growth along the longitudinal axis. Moreover, results from RNAi experiments targeting the contralateral side together with binding studies suggest that homophilic MDGA2 interactions between ipsilaterally projecting axons and post-crossing commissural axons may be the basis of axonal growth along the longitudinal axis. Conclusions: Directed axonal growth of dorsal commissural interneurons requires an elaborate mixture of instructive (guidance) and permissive (outgrowth supporting) molecules. While Wnt and Sonic hedgehog (Shh) signalling pathways have been shown to specify the growth direction of post-crossing commissural axons, our study now provides evidence that homophilic MDGA2 interactions are essential for axonal extension along the longitudinal axis. Interestingly, so far each part of the complex axonal trajectory of commissural axons uses its own set of guidance and growth-promoting molecules, possibly explaining why such a high number of molecules influencing the growth pattern of commissural interneurons has been identified

Expression patterns of plexins and neuropilins are consistent with cooperative and separate functions during neural development

BACKGROUND: Plexins are a family of transmembrane proteins that were shown to act as receptors for Semaphorins either alone or in a complex together with Neuropilins. Based on structural criteria Plexins were subdivided into 4 classes, A through D. PlexinAs are mainly thought to act as mediators of repulsive signals in cell migration and axon guidance. Their functional role in vertebrates has been studied almost exclusively in the context of Semaphorin signaling, i.e. as co-receptors for class 3 Semaphorins. Much less is known about Plexins of the other three classes. Despite the fact that Plexins are involved in the formation of neuronal circuits, the temporal changes of their expression patterns during development of the nervous system have not been analyzed in detail. RESULTS: Only seven plexins are found in the chicken genome in contrast to mammals, where nine plexins have been identified. Here, we describe the dynamic expression patterns of all known plexin family members in comparison to the neuropilins in the developing chicken spinal cord. CONCLUSION: Our in situ hybridization study revealed that the expression patterns of plexins and neuropilins are only partially overlapping, especially during early and intermediate stages of spinal cord development, supporting both cooperative and separate functions of plexins and neuropilins in neural circuit formation

Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system

<p>Abstract</p> <p>Background</p> <p>During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS.</p> <p>Results</p> <p>Downregulation of Sema6A in boundary cap cells by <it>in ovo </it>RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.</p> <p>Conclusion</p> <p>Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.</p

Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner

Upon reaching their intermediate target, the floorplate, commissural axons acquire responsiveness to repulsive guidance cues, allowing the axons to exit the midline and adopt a contralateral, longitudinal trajectory. The molecular mechanisms that regulate this switch from attraction to repulsion remain poorly defined. Here, we show that the heparan sulfate proteoglycan Glypican1 (GPC1) is required as a coreceptor for the Shh-dependent induction of Hedgehog-interacting protein (Hhip) in commissural neurons. In turn, Hhip is required for postcrossing axons to respond to a repulsive anteroposterior Shh gradient. Thus, Shh is a cue with dual function. In precrossing axons it acts as an attractive guidance molecule in a transcription-independent manner. At the same time, Shh binds to GPC1 to induce the expression of its own receptor, Hhip, which mediates the repulsive response of postcrossing axons to Shh. Our study characterizes a molecular mechanism by which navigating axons switch their responsiveness at intermediate targets

The short coiled-coil domain-containing protein UNC-69 cooperates with UNC-76 to regulate axonal outgrowth and normal presynaptic organization in Caenorhabditis elegans

BACKGROUND: The nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however. RESULTS: We show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects. CONCLUSION: We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy