Presenilin-dependent receptor processing is required for axon guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.     

De novo GMP synthesis is required for axon guidance in Drosophila

Guanine nucleotides are key players in mediating growth-cone signaling during neural development. The supply of cellular guanine nucleotides in animals can be achieved via the de novo synthesis and salvage pathways. The de novo synthesis of guanine nucleotides is required for lymphocyte proliferation in animals. Whether the de novo synthesis pathway is essential for any other cellular processes, however, remains unknown. In a search for genes required for the establishment of neuronal connectivity in the fly visual system, we identify the burgundy (bur) gene as an essential player in photoreceptor axon guidance. The bur gene encodes the only GMP synthetase in Drosophila that catalyzes the final reaction of de novo GMP synthesis. Loss of bur causes severe defects in axonal fasciculation, retinotopy, and growth-cone morphology, but does not affect photoreceptor differentiation or retinal patterning. Similar defects were observed when the raspberry (ras) gene, encoding for inosine monophosphate dehydrogenase catalyzing the IMP-to-XMP conversion in GMP de novo synthesis, was mutated. Our study thus provides the first in vivo evidence to support an essential and specific role for de novo synthesis of guanine nucleotides in axon guidance.     

Robo2 is required for Slit-mediated intraretinal axon guidance

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.     

Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling

Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Previous studies showed that Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. Here, we define a seven amino acid sequence in PlexB required for Rac(GTP) binding. The interaction of PlexB with Rac(GTP) is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters Rac(GTP) from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to Rac(GTP) and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA.     

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

The phosphoinositide phosphatase Sac1 is required for midline axon guidance

Sac1 phosphoinositide (PI) phosphatases are important regulators of PtdIns(4)P turnover at the ER, Golgi, and plasma membrane (PM) and are involved in diverse cellular processes including cytoskeletal organization and vesicular trafficking. Here, we present evidence that Sac1 regulates axon guidance in the embryonic CNS of Drosophila. Sac1 is expressed on three longitudinal axon tracts that are defined by the cell adhesion molecule Fasciclin II (Fas II). Mutations in the sac1 gene cause ectopic midline crossing of Fas II-positive axon tracts. This phenotype is rescued by neuronal expression of wild-type Sac1 but not by a catalytically-inactive mutant. Finally, sac1 displays dosage-sensitive genetic interactions with mutations in the genes that encode the midline repellent Slit and its axonal receptor Robo. Taken together, our results suggest that Sac1-mediated regulation of PIs is critical for Slit/Robo-dependent axon repulsion at the CNS midline.     

BMP receptor 1b is required for axon guidance and cell survival in the developing retina

Previous work has documented the importance of BMPs in eye development. Loss-of-function studies in mice, with targeted deletions in either the Bmp7 or Bmp4 genes, have shown that these molecules are critical for early eye development. On the basis of the asymmetry in the dorsal-ventral expression patterns of several members of this family, it has been proposed that these molecules are critical for some aspect of dorsal-ventral patterning in the eye; however, it has been difficult to test this hypothesis because of the early requirement for BMPs in eye development. We have therefore examined the effects of loss of one of the BMP receptors, the BmprIb, on the development of the eye by using targeted deletion. We have found that BmprIb is expressed exclusively in the ventral retina during embryonic development and is required for normal ventral ganglion cell axon targeting to the optic nerve head. In mice with a targeted deletion of the BmprIb gene, many axons arising from the ventrally located ganglion cells fail to enter the optic nerve head, and instead, make abrupt turns in this region. A second phenotype in these mice is a significantly elevated inner retinal apoptosis during a distinct phase of postnatal development, at the end of neurogenesis. Our results therefore show two distinct requirements for BmprIb in mammalian retinal development.     

Epithelial axon guidance in Drosophila

Evidence is offered that the axons of developing sensory neurons in the wing of Drosophila are guided (given both location and polarity information) by the epithelium over which they grow. This guidance is effective in the absence of such potential additional cues as guidepost neurons and physical channels.     

Amalgam is a ligand for the transmembrane receptor neurotactin and is required for neurotactin-mediated cell adhesion and axon fasciculation in Drosophila

Neurotactin (NRT), a member of the cholinesterase-homologous protein family, is a heterophilic cell adhesion molecule that is required for proper axon guidance during Drosophila development. In this study, we identify amalgam (AMA), a member of the immunoglobulin superfamily, as a ligand for the NRT receptor. Using transfected Schneider 2 cells and embryonic primary cultures, we demonstrate that AMA is a secreted protein. Furthermore, AMA is necessary for NRT-expressing cells both to aggregate with themselves and to associate with embryonic primary culture cells. Aggregation assays performed with truncated NRT molecules reveal that the integrity of the cholinesterase-like extracellular domain was not required either for AMA binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the NRT cytoplasmic domain is required for NRT-mediated adhesion, although not for AMA binding. Using an ama-deficient stock, we find that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants.     

Analysis of zebrafish sidetracked mutants reveals a novel role for Plexin A3 in intraspinal motor axon guidance

One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis we show that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, we show that sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, we show that sidetracked (plexin A3) plays an additional role in motor axonal morphogenesis. Together, our data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. We propose that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance.     

Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function

Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. We find that knockin mice expressing a mutant form of EphA4 (EphA4(EE)), whose kinase is constitutively activated in the absence of ephrinB ligands, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 were regulated normally by EphA4(EE), including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation of ephexin1. These results suggest that signaling of Eph RTKs follows a multistep process of induced kinase activity and higher-order clustering different from RTKs responding to soluble ligands.     

G protein-coupled receptor kinase 2 is required for rhythmic olfactory responses in Drosophila

BACKGROUND: The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, we tested whether regulators of ORs were under circadian-clock control. RESULTS: The levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc(01) flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body. CONCLUSIONS: These results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity.     

Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance

Axon guidance receptors guide neuronal growth cones by binding in trans to axon guidance ligands in the developing nervous system. Some ligands are coexpressed in cis with their receptors, raising the question of the relative contribution of cis and trans interactions to axon guidance. Spinal motor axons use Eph receptors to select a limb trajectory in response to trans ephrins, while expressing ephrins in cis. We show that changes in motor neuron ephrin expression result in trajectory selection defects mirrored by changes in growth cone sensitivity to ephrins in vitro, arguing for ephrin cis-attenuation of Eph function. Furthermore, the relative contribution of trans-signaling and cis-attenuation is influenced by the subcellular distribution of ephrins to membrane patches containing Eph receptors. Thus, growth cone ephrins are essential for axon guidance in vivo and the balance between cis and trans modes of axon guidance ligand-receptor interaction contributes to the diversity of axon guidance signaling responses.     

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis

BACKGROUND: Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. RESULTS: Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppressed not only axon pruning but also the infiltration and engulfing action of the surrounding glia. CONCLUSIONS: These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. We therefore propose that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis.